Lactobacillus acidophilus nucleic acid sequences encoding carbohydrate utilization-related proteins and uses therefor

ABSTRACT

Carbohydrate utilization-related and multidrug transporter nucleic acids and polypeptides, and fragments and variants thereof, are disclosed in the current invention. In addition, carbohydrate utilization-related and multidrug transporter fusion proteins, antigenic peptides, and anti-carbohydrate utilization-related and anti-multidrug transporter antibodies are encompassed. The invention also provides vectors containing a nucleic acid of the invention and cells into which the vector has been introduced. Methods for producing the polypeptides and methods of use for the polypeptides of the invention are further disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/903,346, filed Oct. 13, 2010, which is a divisional of U.S. Pat. No.7,838,276, filed Oct. 22, 2008, and which is a divisional of U.S. Pat.No. 7,459,289, filed Mar. 7, 2005, which claims the benefit of U.S.Provisional Application Ser. No. 60/551,121, filed Mar. 8, 2004, thecontents of which are herein incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named396481seqlist.txt, created on Oct. 11, 2010, and having a size of 1,313KB and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to polynucleotides isolated from lactic acidbacteria, namely Lactobacillus acidophilus, and polypeptides encoded bythem, as well as methods for using the polypeptides and organismsexpressing them.

BACKGROUND OF THE INVENTION

Lactobacillus acidophilus is a Gram-positive, rod-shaped, non-sporeforming, homofermentative bacterium that is a normal inhabitant of thegastrointestinal and genitourinary tracts. Since its original isolationby Moro (1900) from infant feces, the “acid loving” organism has beenfound in the intestinal tract of humans, breast-fed infants, and personsconsuming high milk, lactose, or dextrin diets. Historically,Lactobacillus acidophilus is the Lactobacillus species most oftenimplicated as an intestinal probiotic capable of eliciting beneficialeffects on the microflora of the gastrointestinal tract (Klaenhammer andRussell (2000) “Species of the Lactobacillus acidophilus complex,”Encyclopedia of Food Microbiology, 2:1151-1157. Robinson et al., eds.(Academic Press, San Diego, Calif.). Lactobacillus acidophilus canferment hexoses, including lactose and more complex oligosaccharides, toproduce lactic acid and lower the pH of the environment where theorganism is cultured. Acidified environments (e.g., food, vagina, andregions within the gastrointestinal tract) can interfere with the growthof undesirable bacteria, pathogens, and yeasts. The organism is wellknown for its acid tolerance, survival in cultured dairy products, andviability during passage through the stomach and gastrointestinal tract.Lactobacilli and other commensal bacteria, some of which are consideredprobiotic bacteria that “favor life,” have been studied extensively fortheir effects on human health, particularly in the prevention ortreatment of enteric infections, diarrheal disease, prevention ofcancer, and stimulation of the immune system. Lactobacilli have alsobeen studied for their influence on dairy product flavor, and functionaland textural characteristics. Genetic characterization of otherLactobacillus species (e.g., L johnsonii and L. rhamnosus) has beendescribed (see e.g., U.S. Pat. No. 6,476,209; U.S. Pat. No. 6,544,772;U.S. Patent Publication Nos. 20020159976, 2003013882 & 20040009490; PCTPublication No. WO 2004/031389; PCT Publication No. 2003/084989; PCTPublication No. WO 2004/020467).

Bacterial growth requires specific transport systems to import nutrientsfrom the external environment. Lactic acid bacteria transport moleculesinto and out of the cell via three systems: primary transport, secondarytransport, and group translocation. In primary transport, chemical(primarily ATP), electrical, or solar energy is used to drive transport.ATP-binding cassette (ABC) transporters are the most abundant class ofprimary transport systems in lactic acid bacteria. In this system, ATPhydrolysis is linked with substrate translocation across the membranefor both the import of sugars and compatible solutes and the export ofproducts such as drugs or toxins that are undesirable to the cell, orcellular components that function outside of the cell, such as cell wallpolysaccharides. In general, ABC transporters are relatively specificfor their substrates, but some are multispecific, such as the multidrugtransporters.

Secondary transport systems use electrochemical gradients to provide theenergy for sugar translocation. They comprise symporters, whichcotransport two or more solutes, uniporters, which transport onemolecule, and antiporters, which countertransport two or more solutes.Symporters generally couple the uphill movement of the substrate to thedownhill movement of a proton (or ion), antiporters use the ion gradientfor excretion of a product, and uniporters do not use a coupling ion(Poolman (2002) Anionic van Leeuwenhoek 82:147-164).

Group translocation involves the phosphoenolpyruvate (PEP)-dependentphosphotransferase system (PTS), which couples the uptake of acarbohydrate or alditol with its phosphorylation (Poolman (2002),supra). The phosphate group originates from the conversion of PEP intopyruvate, and the subsequent phosphorylation involves the energycoupling proteins, Enzyme I and HPr, as well as substrate-specificphosphoryl transfer proteins IIA, IIB and IIC.

Multidrug transporters may be separated into two major classes,secondary multidrug transporters and ABC transporters. Secondarymultidrug transporters may be further divided into distinct families,including the major facilitator superfamily (MFS), the small multidrugresistance family (SMR), the resistance-nodulation-cell division family(RND), and the multidrug and toxic compound extrusion family (MATE)(Putman et al. (2000) Microbiol. Mol. Biol. Reviews 64:672-693).Secondary multidrug transporters use the electrochemical gradients, asdescribed herein, to extrude drugs from the cell. ABC-type multidrugtransporters use energy from ATP hydrolysis to pump drugs out of thecell (Putman et al. (2000), supra).

Bacteria are able to metabolize various carbohydrates by utilizingtransport proteins and enzymes with different carbohydratespecificities, in addition to employing diverse regulatory mechanisms,such as catabolite repression. The isolation and characterization ofthese proteins allows for the development of essential probioticproducts with numerous applications, including those that benefit humanand/or animal health, and those concerned with food production andsafety. The proteins can also be used in developing transgenic plantswith altered growth or survival capabilities.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for modifying microorganisms and plants areprovided. Compositions of the invention include isolated nucleic acidsfrom Lactobacillus acidophilus encoding carbohydrate utilization-relatedproteins, including proteins of the phosphotransferase system (PTS), ABCtransporters, and other proteins involved in transport, degradation,and/or synthesis of sugars in Lactobacillus acidophilus. Compositionsalso include isolated nucleic acids from Lactobacillus acidophilus thatencode multidrug transporters. Specifically, the present inventionprovides isolated nucleic acid molecules comprising, consistingessentially of and/or consisting of the nucleotide sequence as set forthin odd numbered SEQ ID NOS:1-321, singly and/or in any combination, andisolated nucleic acid molecules encoding the amino acid sequence as setforth found in even numbered SEQ ID NOS:2-322, singly and/or in anycombination. Also provided are isolated and/or recombinant polypeptidescomprising, consisting essentially of and/or consisting of an amino acidsequence encoded by a nucleic acid molecule described herein and/or asset forth in even numbered SEQ ID NOS:2-322, singly and/or in anycombination. Variant nucleic acids and polypeptides sufficientlyidentical to the nucleotide sequences and amino acid sequences set forthin the Sequence Listing are encompassed by the present invention.Additionally, fragments and sufficiently identical fragments of thenucleotide sequences and amino acid sequences are encompassed.Nucleotide sequences that are complementary to a nucleic acid sequenceof the invention, or that hybridize to a nucleotide sequence of theinvention, are also encompassed.

Compositions further include vectors and prokaryotic, eukaryotic andplant cells for recombinant expression of the nucleic acids describedherein, as well as transgenic microbial and plant populations comprisingthe vectors. Also included in the invention are methods for therecombinant production of the polypeptides of the invention, and methodsfor their use. Further included are methods and kits for detecting thepresence of a nucleic acid and/or polypeptide sequence of the inventionin a sample, and antibodies that bind to a polypeptide of the invention.

The carbohydrate utilization-related and multidrug transporter moleculesof the present invention are useful for the selection and production ofrecombinant bacteria, particularly the production of bacteria withimproved fermentative abilities. Such bacteria include, but are notlimited to, bacteria that have a modified ability to synthesize,transport, accumulate, and/or utilize various carbohydrates, bacteriawith altered flavors or textures, bacteria that produce alteredcarbohydrates, and bacteria better able to survive stressful conditions,such as those encountered in food processing and/or in thegastrointestinal tract of an animal. The multidrug transporter moleculesof the present invention include those that allow bacteria to bettersurvive contact with antimicrobial polypeptides, such as bacteriocins orother toxins. These carbohydrate utilization-related and multidrugtransporter molecules are also useful for modifying plant species.Transgenic plants comprising one or more sequences of the presentinvention may be beneficial economically in that they are moreresistance to environmental stresses, including, but not limited to,plant pathogens, high salt concentration, or dehydration. They may alsobe better able to withstand food processing and storage conditions.

The present invention provides an isolated nucleic acid selected fromthe group consisting of a nucleic acid comprising, consisting of and/orconsisting essentially of a nucleotide sequence as set forth in SEQ IDNOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301,303, 305, 307, 309, 311, 313, 315, 317, 319 and/or 321 in anycombination, including multiples of the same sequence, and/or acomplement thereof, a nucleic acid comprising, consisting of and/orconsisting essentially of a nucleotide sequence having at least 90%sequence identity to a nucleotide sequence as set forth in SEQ ID NO:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 313, 315, 317, 319 and/or 321 in any combination,including multiples of the same sequence, and/or a complement thereof, anucleic acid comprising, consisting of and/or consisting essentially ofa fragment of a nucleotide sequence as set forth in SEQ ID NO:1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 313, 315, 317, 319 and/or 321 in any combination,including multiples of the same sequence, and/or a complement thereof, anucleic acid that encodes a polypeptide comprising an amino acidsequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,320 and/or 322 in any combination, including multiples of the samesequence, and/or encoded by a nucleic acid molecule described herein, anucleic acid comprising a nucleotide sequence encoding a polypeptidehaving at least 90% amino acid sequence identity to the amino acidsequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,320 and/or 322 in any combination, including multiples of the samesequence, and/or encoded by a nucleic acid molecule described herein,and a nucleic acid that hybridizes under stringent conditions to any ofthe above.

Compositions further include vectors comprising the nucleic acidsdescribed herein, vectors further comprising a nucleic acid encoding aheterologous polypeptide, and cells, including bacterial, plant andeukaryotic cells, containing said vectors. Also included in theinvention are methods for the recombinant production of the polypeptidesof the invention, and methods for their use. Further included aremethods and kits for detecting the presence of a nucleic acid orpolypeptide sequence of the invention in a sample, and antibodies thatbind to a polypeptide of the invention.

The present invention further provides an isolated polypeptide selectedfrom the group consisting of: a) a polypeptide comprising, consisting ofand/or consisting essentially of an amino acid sequence as set forth inSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320 and/or 322 in anycombination, including multiples of the same sequence, and/or encoded bya nucleic acid molecule described herein; b) a polypeptide comprising,consisting of and/or consisting essentially of a fragment of an aminoacid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,318, 320 and/or 322 in any combination, including multiples of the samesequence, and/or encoded by a nucleic acid molecule described herein; c)a polypeptide comprising, consisting of and/or consisting essentially ofan amino acid sequence having at least 90% sequence identity with anamino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320 and/or 322 in any combination, including multiples ofthe same sequence, and/or encoded by a nucleic acid molecule describedherein; d) a polypeptide encoded by a nucleotide sequence having atleast 90% sequence identity to a nucleotide sequence as set forth in SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301,303, 305, 307, 309, 311, 313, 315, 317, 319 and/or 321 in anycombination; and e) a polypeptide encoded by a nucleotide sequence asset forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319 and/or321 in any combination.

Also provided is a polypeptide of this invention further comprising oneor more heterologous amino acid sequences, and antibodies thatselectively bind to the polypeptides described herein.

Additionally provided are methods for producing a polypeptide, saidmethod comprising culturing the cell of this invention under conditionsin which a nucleic acid encoding the polypeptide is expressed, saidpolypeptide being selected from the group consisting of: a) apolypeptide comprising an amino acid sequence of SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, 316, 318, 320 and/or 322 in any combination, includingmultiples of the same sequence, and/or encoded by a nucleic acidmolecule described herein; b) a polypeptide comprising a fragment of anamino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320 and/or 322 in any combination, including multiples ofthe same sequence, and/or encoded by a nucleic acid molecule describedherein; c) a polypeptide comprising an amino acid sequence having atleast 90% sequence identity with an amino acid sequence as set forth inSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320 and/or 322 in anycombination, including multiples of the same sequence, and/or encoded bya nucleic acid molecule described herein; d) a polypeptide encoded by anucleotide sequence having at least 90% sequence identity to anucleotide sequence as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319 and/or 321 in any combination; and e) a polypeptideencoded by a nucleotide sequence as set forth in SEQ ID NO:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319 and/or 321 in any combination.

Also provided are methods for detecting the presence of a polypeptide ina sample, said method comprising contacting the sample with a compoundthat selectively binds to a polypeptide and determining whether thecompound binds to the polypeptide in the sample; wherein saidpolypeptide is selected from the group consisting of: a) a polypeptideencoded by a nucleotide sequence as set forth in SEQ ID NO:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319 and/or 321 in any combination; b) apolypeptide comprising a fragment of an amino acid sequence encoded by anucleic acid sequence as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319 and/or 321 in any combination; c) a polypeptide encoded bya nucleotide sequence having at least 90% sequence identity to anucleotide sequence as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319 and/or 321 in any combination; d) a polypeptide comprisingan amino acid sequence having at least 90% sequence identity to an aminoacid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,318, 320 and/or 322 in any combination; and e) a polypeptide comprisingan amino acid sequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320 and/or 322 in any combination.

Additionally provided are methods for detecting the presence of apolypeptide in a sample, said method comprising contacting the samplewith a compound that selectively binds to a polypeptide and determiningwhether the compound binds to the polypeptide in the sample of theinvention, wherein the compound that binds to the polypeptide is anantibody. Also provided is a kit comprising a compound for use in themethods of the invention and instructions for use.

The present invention also provides methods for detecting the presenceof a nucleic acid molecule and/or fragments thereof of this invention ina sample, comprising: a) contacting the sample with a nucleic acid probeor primer that selectively hybridizes to the nucleic acid moleculeand/or fragment thereof; and b) determining whether the nucleic acidprobe or primer hybridizes to a nucleic acid molecule in the sample,thereby detecting the presence of a nucleic acid molecule and/orfragment thereof of this invention in the sample. Also provided aremethods for detecting the presence of a nucleic acid molecule and/orfragment of the invention in a sample wherein the sample comprises mRNAmolecules and is contacted with a nucleic acid probe. Additionallyprovided herein is a kit comprising a compound that selectivelyhybridizes to a nucleic acid of the invention, and instructions for use.

Additionally provided are methods for 1) modifying the ability of anorganism to transport a carbohydrate into or out of a cell; 2) modifyingthe ability of an organism to accumulate a carbohydrate; 3) modifyingthe ability of an organism to utilize a carbohydrate as an energysource; 4) modifying the ability of an organism to produce a modifiedcarbohydrate; 5) modifying the flavor of a food product fermented by amicroorganism; 6) modifying the texture of a food product fermented by amicroorganism; 7) modifying the ability of an organism to survive foodprocessing and storage conditions; 8) modifying the ability of amicroorganism to survive in a gastrointestinal (GI) tract; 9) modifyingthe ability of an organism to transport a drug into or out of a cell;and 10) modifying the ability of an organism to produce a carbohydrate,comprising introducing into said organism and/or microorganism a vectorcomprising at least one nucleotide sequence of this invention and/or atleast one nucleotide sequence selected from the group consisting of: a)a nucleotide sequence as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319 and/or 321 in any combination; b) a nucleotide sequencecomprising a fragment of a nucleotide sequence as set forth in SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301,303, 305, 307, 309, 311, 313, 315, 317, 319 and/or 321 in anycombination, wherein said fragment encodes a polypeptide that retainsactivity; c) a nucleotide sequence that is at least 90% identical to thesequence as set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,319 and/or 321 in any combination, wherein said nucleotide sequenceencodes a polypeptide that retains activity; and d) a nucleotidesequence encoding a polypeptide comprising an amino acid sequence havingat least 90% sequence identity to an amino acid sequence as set forth inSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320 and/or 322 in anycombination, wherein said polypeptide retains activity; and e) anucleotide sequence encoding a polypeptide comprising an amino acidsequence as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,320 and/or 322 in any combination.

Further provided herein is 1) a Lactobacillus acidophilus bacterialstrain with a modified ability to transport a carbohydrate into or outof a cell as compared to a wild-type Lactobacillus acidophilus; 2) aLactobacillus acidophilus bacterial strain with a modified ability toaccumulate a carbohydrate, as compared to a wild-type Lactobacillusacidophilus; 3) a Lactobacillus acidophilus bacterial strain with amodified ability to utilize a carbohydrate as an energy source, ascompared to a wild-type Lactobacillus acidophilus; 4) a Lactobacillusacidophilus bacterial strain that provides a food product with amodified flavor as a result of fermentation, as compared to a wild-typeLactobacillus acidophilus; 5) a Lactobacillus acidophilus bacterialstrain that provides a food product with a modified texture as a resultof fermentation, as compared to a wild-type Lactobacillus acidophilus;6) a Lactobacillus acidophilus bacterial strain with a modified abilityto produce a carbohydrate, as compared to a wild-type Lactobacillusacidophilus; 7) a Lactobacillus acidophilus bacterial strain with amodified ability to survive food processing and storage conditions, ascompared to a wild-type Lactobacillus acidophilus; and 8) aLactobacillus acidophilus bacterial strain with a modified ability tosurvive in a GI tract, as compared to a wild-type Lactobacillusacidophilus, wherein said modified ability, flavor and/or texture is dueto expression of at least one carbohydrate utilization-relatedpolypeptide as set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,318, 320 and/or 322 in any combination.

Additionally provided is a Lactobacillus acidophilus bacterial strainwith a modified ability to survive contact with an antimicrobialpolypeptide or toxin, as compared to a wild-type Lactobacillusacidophilus, wherein said modified ability is due to expression of atleast one multidrug transport polypeptide as set forth in even SEQ IDNOs:78-88, 92-94, 124-126, 132, 282-288, 308 and/or 312-322.

Also provided is a plant, a plant cell and/or a seed of a plant, havingstably incorporated into its genome a DNA construct comprising at leastone nucleotide sequence of this invention and/or at least one nucleotidesequence of this invention, selected from the group consisting of: a) anucleotide sequence as set forth in any of SEQ ID NOs:1-321, singlyand/or in any combination, or a complement thereof; b) a nucleotidesequence having at least 90% sequence identity to a nucleotide sequenceas set forth in any of SEQ ID NOs:1-321, singly and/or in anycombination, or a complement thereof; c) a nucleotide sequencecomprising a fragment of a nucleotide sequence as set forth in any ofSEQ ID NOs:1-321, singly and/or in any combination, or a complementthereof; d) a nucleotide sequence that encodes a polypeptide comprisingan amino acid sequence as set forth in any of SEQ ID NOs:2-322; e) anucleotide sequence that encodes a polypeptide comprising an amino acidsequence having at least 90% sequence identity to the amino acidsequence as set forth in any of SEQ ID NOs:2-322 and f) a nucleotidesequence that hybridizes under stringent conditions to any of a)-e).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Genetic loci of interest. The layouts of the loci discussed inthe text are shown: man, glucose-mannose locus; fru, fructose locus;suc, sucrose locus; Jos, FOS locus; raft; raffinose locus; Lac,lactose-galactose loci; tre, trehalose locus; CCR, carbon cataboliteloci.

FIG. 2. Carbohydrate utilization in Lactobacillus acidophilus. Thisdiagram shows carbohydrate transporters and hydrolases as predicted bytranscriptional profiles. Protein names and EC numbers are specified foreach element. PTS transporters are shown in black. GPH transporters areshown in light gray. ABC transporters are shown in dark gray.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to carbohydrate utilization-related andmultidrug transport molecules from Lactobacillus acidophilus. Nucleotideand amino acid sequences of the carbohydrate utilization-related andmultidrug transport molecules are provided. The sequences are useful formodifying microorganisms, cells and plants for enhanced properties.

As used herein, “a,” “an” and “the” can be plural or singular as usedthroughout the specification and claims. For example “a” cell can mean asingle cell or a multiplicity of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

By “carbohydrate utilization-related” molecules or genes is meant novelsequences from Lactobacillus acidophilus that encode proteins involvedin the utilization of carbohydrate molecules, including, but not limitedto, the synthesis, transport, or degradation of carbohydrates. By“multidrug transporter” molecules is meant those that are involved inthe transport of antimicrobial polypeptides such as bacteriocins, orother drugs or toxins. See Table 1 for specific carbohydrateutilization-related and multidrug transporter molecules of the presentinvention. The full-length gene sequences are referred to as“carbohydrate utilization-related sequences” or “multidrug transportersequences,” showing that they have similarity to carbohydrateutilization-related genes or multidrug transporter genes, respectively.The invention further provides fragments and variants of thesecarbohydrate utilization related sequences or multidrug transportersequences, which can also be used to practice methods of the presentinvention.

By “carbohydrate” is meant an organic compound containing carbon,hydrogen, and oxygen, usually in the ratio 1:2:1. Carbohydrates include,but are not limited to, sugars, starches, celluloses, and gums. As usedherein, the terms “gene” and “recombinant gene” refer to nucleic acidscomprising an open reading frame, particularly those encoding acarbohydrate utilization-related protein or a multidrug transporterprotein. Isolated nucleic acids of the present invention comprisenucleic acid sequences encoding carbohydrate utilization-relatedproteins or multidrug transporter proteins, nucleic acid sequencesencoding the amino acid sequences set forth in even numbered SEQ IDNOS:2-322, the nucleic acid sequences set forth in odd numbered SEQ IDNOS:1-321, and variants and fragments thereof. The present inventionalso encompasses antisense nucleic acids, as described below.

In addition, isolated polypeptides and proteins having carbohydrateutilization-related activity or multidrug transporter activity, andvariants and fragments thereof, are encompassed, as well as methods forproducing those polypeptides. For purposes of the present invention, theterms “protein” and “polypeptide” are used interchangeably. Thepolypeptides of the present invention have carbohydrateutilization-related protein activity or multidrug transporter activity.Carbohydrate utilization-related protein activity or multidrugtransporter activity refers to a biological or functional activity asdetermined in vivo or in vitro according to standard assay techniques.These activities include, but are not limited to, the ability tosynthesize a carbohydrate, the ability to transport a carbohydrate intoor out of a cell, the ability to degrade a carbohydrate, the ability toregulate the concentration of a carbohydrate in a cell, the ability tobind a carbohydrate, and the ability to transport a drug or toxin intoor out of a cell.

The structures of the various types of bacterial transporters are wellknown in the art. The ATP-binding cassette (ABC) superfamily oftransporters consists of proteins with four core domains (Higgins et al.(1986) Nature 323:448-450; Hyde et al. (1990) Nature 346:362-365;Higgins (2001) Res. Microbiol. 152:205-210). Typically there are twotransmembrane domains with six membrane-spanning alpha helices perdomain, and two ATP-binding domains that contain the core amino acids bywhich the transporters are defined (Higgins (2001) supra.), as well asthe other conserved motifs including the Walker A and Walker B motifs(Walker et al. (1982) EMBO J. 1:945-951; Prosite Ref. No. PDO000185).

The secondary transport system proteins include thegalactoside-pentose-hexuronide group of translocators (Poolman et al.(1996) Mol. Microbiol. 19:911-922). These proteins generally consist ofa hydrophobic domain comprising twelve membrane spanning domains and acarboxyterminal enzyme IIA domain (Poolman et al. (1989) J. Bacteriol.171:244-253).

The phosphotransferase system (PTS) proteins include enzyme-I (PrositeRef. No. PDO000527), the phosphoryl carrier proteins (HPr) (Prosite Ref.No. PDO000318), and the sugar-specific permease, which consists of atleast three structurally distinct domains (Prosite Ref. Nos. PDO000528;PDO000795). The HPr protein contains two conserved phosphorylationsites, a histidine residue at the amino-terminal side that isphosphorylated by Enzyme I, and a serine residue at the carboxy-terminalside of the protein that may be phosphorylated by an ATP-dependentprotein kinase (de Vos (1996) Anionic van Leeuwenhoek 70:223-242).

Members of the major facilitator super family (MFS) of multidrugtransporters have either 12 or 14 transmembrane segments. Members of thesmall multidrug resistance family (SMR) of multidrug transporters arethought to form a tightly packed four-helix antiparallel bundle. Membersof the resistance nodulation-cell division family (RND) contain a singleN-terminal transmembrane segment and a large C-terminal periplasmicdomain (Putman et al. (2000) Microbiol. Mol. Biol. Reviews 64:672-693).Conserved motifs within each of these types of multidrug transportersand also throughout the multidrug transporters of the MFS, SMR, and RNDfamilies, as well as specific proteins from various bacteria (withAccession Nos.) have been described (Putman et al. (2000) supra).

The nucleic acid and protein compositions encompassed by the presentinvention are isolated or substantially purified. By “isolated” or“substantially purified” is meant that the nucleic acid or proteinmolecules, or biologically active fragments or variants thereof, aresubstantially or essentially free from components normally found inassociation with the nucleic acid or protein in its natural state. Suchcomponents include other cellular material, culture medium fromrecombinant production, and/or various chemicals used in chemicallysynthesizing the proteins or nucleic acids. Preferably, an “isolated”nucleic acid of the present invention is free of nucleic acid sequencesthat flank the nucleic acid of interest in the genomic DNA of theorganism from which the nucleic acid was obtained (such as codingsequences present at the 5′ or 3′ ends). However, the molecule mayinclude some additional bases or moieties that do not deleteriouslyaffect the basic characteristics of the composition. For example, invarious embodiments, the isolated nucleic acid contains less than 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequencenormally associated with the genomic DNA in the cells from which it wasobtained. Similarly, a substantially purified protein has less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein,or non-carbohydrate utilization-related protein. When the protein isrecombinantly produced, preferably culture medium represents less than30%, 20%, 10%, or 5% of the volume of the protein preparation, and whenthe protein is produced chemically, preferably the preparations haveless than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors, or non-carbohydrate utilization-related chemicals.

The compositions and methods of the present invention can be used tomodulate the function of the carbohydrate utilization-related ormultidrug transporter molecules of Lactobacillus acidophilus. By“modulate,” “alter,” or “modify” is meant the up- or downregulation of atarget biological activity. Proteins of the invention are useful inmodifying the biological activities of lactic acid bacteria, and also inmodifying the nutritional or health-promoting characteristics of foodsfermented by such bacteria. Nucleotide molecules of the invention areuseful in modulating carbohydrate utilization-related or multidrugtransporter protein expression by lactic acid bacteria. Up- ordownregulation of expression from a nucleic acid of the presentinvention is encompassed. Upregulation may be accomplished, for example,by providing multiple gene copies, modulating expression by modifyingregulatory elements, promoting transcriptional or translationalmechanisms, or other means. Downregulation may be accomplished, forexample, by using known antisense and gene silencing techniques.

By “lactic acid bacteria” is meant bacteria from a genus selected fromthe following: Aerococcus, Carnobacterium, Enterococcus, Lactococcus,Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus,Melissococcus, Alloiococcus, Dolosigranulum, Lactosphaera,Tetragenococcus, Vagococcus, and Weissella (Holzapfel et al. (2001)Am.J. Clin. Nutr. 73:365 S-373S; Bergey's Manual of SystematicBacteriology, Vol. 2 (Williams and Wilkins, Baltimore; (1986)) pp.1075-1079).

The polypeptides of the present invention or microbes expressing themare useful as nutritional additives or supplements, and as additives indairy and fermentation processing. The nucleic acid sequences, encodedpolypeptides, and microorganisms expressing them are useful in themanufacture of milk-derived products, such as cheeses, yogurt, fermentedmilk products, sour milks, and buttermilk. Microorganisms that expresspolypeptides of the invention may be probiotic organisms. By “probiotic”is meant a live microorganism that survives passage through thegastrointestinal tract and has a beneficial effect on the subject. By“subject” is meant an organism that comes into contact with amicroorganism expressing a protein of the present invention. Subject mayrefer to humans and other animals.

In addition to the carbohydrate utilization-related and multidrugtransporter nucleotide sequences and fragments and variants thereof asdisclosed herein, the nucleic acids of the current invention alsoencompass homologous nucleic acid sequences identified and isolated fromother organisms or cells by hybridization with entire or partialsequences obtained from the carbohydrate utilization-related andmultidrug transporter nucleotide sequences or variants and fragmentsthereof as disclosed herein.

Fragments and Variants

The invention provides isolated nucleic acids comprising nucleotidesequences encoding carbohydrate utilization-related and multidrugtransporter proteins, as well as the carbohydrate utilization-relatedand multidrug transporter proteins encoded thereby. By “carbohydrateutilization-related protein” is meant a protein having an amino acidsequence as set forth in even numbered SEQ ID NOS:2-322. Fragments andvariants of these nucleotide sequences and encoded proteins are alsoprovided. By “fragment” of a nucleotide sequence or protein is meant aportion of the nucleotide or amino acid sequence.

Fragments of the nucleic acids disclosed herein can be used ashybridization probes to identify carbohydrateutilization-related-encoding nucleic acids or multidrugtransporter-encoding nucleic acids, or can be used as primers inamplification protocols [e.g., polymerase chain reaction (PCR)] ormutation of carbohydrate utilization-related or multidrug transporternucleic acids. Fragments of nucleic acids of this invention can also bebound to a physical substrate to comprise what may be considered amacro- or microarray (see, for example, U.S. Pat. No. 5,837,832; U.S.Pat. No. 5,861,242; WO 89/10977; WO 89/11548; WO 93/17126; U.S. Pat. No.6,309,823). Such arrays or “chips” of nucleic acids may be used to studygene expression or to identify nucleic acids with sufficient identity tothe target sequences.

The present invention further provides a nucleic acid array or chip,i.e., a multitude of nucleic acids (e.g., DNA) as molecular probesprecisely organized or arrayed on a solid support, which allow for thesequencing of genes, the study of mutations contained therein and/or theanalysis of the expression of genes, as such arrays and chips arecurrently of interest given their very small size and their highcapacity in terms of number of analyses.

The function of these nucleic acid arrays/chips is based on molecularprobes, mainly oligonucleotides, which are attached to a carrier havinga size of generally a few square centimeters or more, as desired. For ananalysis, the carrier, such as in a DNA array/chip, is coated with DNAprobes (e.g., oligonucleotides) that are arranged at a predeterminedlocation or position on the carrier. A sample containing a targetnucleic acid and/or fragments thereof to be analyzed, for example DNA orRNA or cDNA, that has been labeled beforehand, is contacted with the DNAarray/chip leading to the formation, through hybridization, of a duplex.After a washing step, analysis of the surface of the chip allows anyhybridizations to be located by means of the signals emitted by thelabeled target. A hybridization fingerprint results, which, by computerprocessing, allows retrieval of information such as the expression ofgenes, the presence of specific fragments in the sample, thedetermination of sequences and/or the identification of mutations.

In one embodiment of this invention, hybridization between targetnucleic acids and nucleic acids of the invention, used in the form ofprobes and deposited or synthesized in situ on a DNA chip/array, can bedetermined by means of fluorescence, radioactivity, electronic detectionor the like, as are well known in the art.

In another embodiment, the nucleotide sequences of the invention can beused in the form of a DNA array/chip to carry out analyses of theexpression of Lactobacillus acidophilus genes. This analysis is based onDNA array/chips on which probes, chosen for their specificity tocharacterize a given gene or nucleotide sequence, are present. Thetarget sequences to be analyzed are labeled before being hybridized ontothe chip. After washing, the labeled complexes are detected andquantified, with the hybridizations being carried out at least induplicate. Comparative analyses of the signal intensities obtained withrespect to the same probe for different samples and/or for differentprobes with the same sample, allows, for example, for differentialtranscription of RNA derived from the sample.

In yet another embodiment, arrays/chips containing nucleotide sequencesof the invention can comprise nucleotide sequences specific for othermicroorganisms, which allows for serial testing and rapid identificationof the presence of a microorganism in a sample.

In a further embodiment, the principle of the DNA array/chip can also beused to produce protein arrays/chips on which the support has beencoated with a polypeptide and/or an antibody of this invention, orarrays thereof, in place of the nucleic acid. These protein arrays/chipsmake it possible, for example, to analyze the biomolecular interactionsinduced by the affinity capture of targets onto a support coated, e.g.,with proteins, by surface plasma resonance (SPR). The polypeptides orantibodies of this invention, capable of specifically binding antibodiesor polypeptides derived from the sample to be analyzed, can be used inprotein arrays/chips for the detection and/or identification of proteinsand/or peptides in a sample.

Thus, the present invention provides a microarray or microchipcomprising various nucleic acids of this invention in any combination,including repeats, as well as a microarray comprising variouspolypeptides of this invention in any combination, including repeats.Also provided is a microarray comprising antibodies that specificallyreact with various polypeptides of this invention, in any combination,including repeats.

By “nucleic acid” is meant DNA molecules (e.g., cDNA or genomic DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid can be single-stranded ordouble-stranded, but is typically double-stranded DNA. A fragment of anucleic acid encoding a carbohydrate utilization-related protein or amultidrug transporter protein may encode a protein fragment that isbiologically active, or it may be used as a hybridization probe or PCRprimer as described herein. A biologically active fragment of apolypeptide disclosed herein can be prepared by isolating a portion ofone of the nucleotide sequences of the invention, expressing the encodedportion of the protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the protein. Fragmentsof nucleic acids encoding carbohydrate utilization-related or multidrugtransporter proteins comprise at least about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 75, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2200, or 2500contiguous nucleotides, including any number between 5 and 2500 notspecifically recited herein, or up to the total number of nucleotidespresent in a full-length carbohydrate utilization-related or multidrugtransporter nucleotide sequence as disclosed herein (for example, 432for SEQ ID NO:1, 369 for SEQ ID NO:3, etc.).

Fragments of amino acid sequences include polypeptide fragments suitablefor use as immunogens to raise anti-carbohydrate utilization-related oranti-multidrug transporter antibodies. Fragments include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of a carbohydrate utilization-related ormultidrug transporter protein, or partial-length protein, of theinvention and exhibiting at least one activity of a carbohydrateutilization-related or multidrug transporter protein, but which includefewer amino acids than the full-length proteins disclosed herein.Typically, biologically active portions comprise a domain or motif withat least one activity of the carbohydrate utilization-related ormultidrug transporter protein. A biologically active portion of acarbohydrate utilization-related or multidrug transporter protein can bea polypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650 contiguous amino acids in length, orany number between 10 and 650 not specifically recited herein, up to thetotal number of amino acids present in a full-length protein of thecurrent invention (for example, 144 for SEQ ID NO:2, 123 for SEQ IDNO:4, etc.). Such biologically active portions can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native carbohydrate utilization-related or multidrugtransporter protein. As used here, a fragment comprises at least 5contiguous amino acids of any of even numbered SEQ ID NOS:2-322. Theinvention encompasses other fragments, however, such as any fragment inthe protein greater than 6, 7, 8, or 9 amino acids.

Variants of the nucleotide and amino acid sequences are encompassed inthe present invention. By “variant” is meant a sufficiently identicalsequence. Accordingly, the invention encompasses isolated nucleic acidsthat are sufficiently identical to the nucleotide sequences encodingcarbohydrate utilization-related proteins and multidrug transporterproteins in even numbered SEQ ID NOS:2-322, or nucleic acids thathybridize to a nucleic acid of odd numbered SEQ ID NOS:1-321, or acomplement thereof, under stringent conditions. Variants also includepolypeptides encoded by the variant nucleotide sequences of the presentinvention. In addition, polypeptides of the current invention have anamino acid sequence that is sufficiently identical to an amino acidsequence set forth in even numbered SEQ ID NOS:1-320. By “sufficientlyidentical” is meant that a first amino acid or nucleotide sequencecontains a sufficient or minimal number of equivalent or identical aminoacid residues as compared to a second amino acid or nucleotide sequence,thus providing a common structural domain and/or indicating a commonfunctional activity. Conservative variants include those sequences thatdiffer due to the degeneracy of the genetic code.

In general, amino acid or nucleotide sequences that have at least about45%, 55%, or 65% identity, preferably at least about 70% or 75%identity, more preferably at least about 80%, 85% or 90%, mostpreferably at least about 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%,or 99% sequence identity to any of the amino acid sequences of evennumbered SEQ ID NOS:2-322 or any of the nucleotide sequences of oddnumbered SEQ ID NOS:1-321, respectively, are defined herein assufficiently identical. Variant proteins encompassed by the presentinvention are biologically active, that is they retain the desiredbiological activity of the native protein, that is, carbohydrateutilization-related activity or multidrug transporter activity asdescribed herein. A biologically active variant of a protein of theinvention may differ from that protein by as few as 1-15 amino acidresidues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

Naturally occurring variants may exist within a population (e.g., theLactobacillus acidophilus population). Such variants can be identifiedby using well-known molecular biology techniques, such as the polymerasechain reaction (PCR), and hybridization as described below.Synthetically derived nucleotide sequences, for example, sequencesgenerated by site-directed mutagenesis or PCR-mediated mutagenesis, thatstill encode a carbohydrate utilization-related protein or multidrugtransporter protein, are also included as variants. One or morenucleotide or amino acid substitutions, additions, or deletions can beintroduced into a nucleotide or amino acid sequence disclosed herein,such that the substitutions, additions, or deletions are introduced intothe encoded protein. The additions (insertions) or deletions(truncations) may be made at the N-terminal or C-terminal end of thenative protein, or at one or more sites in the native protein.Similarly, a substitution of one or more nucleotides or amino acids maybe made at one or more sites in the native protein.

For example, conservative amino acid substitutions may be made at one ormore predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a protein without altering the biologicalactivity, whereas an “essential” amino acid is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue with a similarside chain. Families of amino acid residues having similar side chainsare known in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Suchsubstitutions would not be made for conserved amino acid residues, orfor amino acid residues residing within a conserved motif, where suchresidues are essential for protein activity.

Alternatively, mutations can be made randomly along all or part of thelength of the carbohydrate utilization-related or multidrug transportercoding sequence, such as by saturation mutagenesis. The mutants can beexpressed recombinantly, and screened for those that retain biologicalactivity by assaying for carbohydrate utilization-related or multidrugtransporter activity using standard assay techniques. Methods formutagenesis and nucleotide sequence alterations are known in the art.See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;Kunkel et al. (1987) Methods in Enzymol. Molecular Biology (MacMillanPublishing Company, New York) and the references sited therein.Obviously the mutations made in the DNA encoding the variant must notdisrupt the reading frame and preferably will not create complementaryregions that could produce secondary mRNA structure. See, EP PatentApplication Publication No. 75,444. Guidance as to appropriate aminoacid substitutions that do not effect biological activity of the proteinof interest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by comparing the activity of the modified sequence with theactivity of the original sequence. See the “Methods of Use” sectionbelow for examples of assays that may be used to measure carbohydrateutilization-related activity or multidrug transporter activity.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent carbohydrate utilization-related or multidrug transporterprotein coding regions can be used to create a new carbohydrateutilization-related protein or a new multidrug transporter proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the carbohydrateutilization-related or multidrug transporter gene of the invention andother known carbohydrate utilization-related or multidrug transportergenes to obtain a new gene coding for a protein with an improvedproperty of interest, such as an increased K_(m) in the case of anenzyme. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Variants of the carbohydrate utilization-related and multidrugtransporter proteins can function as either agonists (mimetics) or asantagonists. An agonist of the protein can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the protein. An antagonist of the protein can inhibitone or more of the activities of the naturally occurring form of theprotein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade that includes thecarbohydrate utilization-related or multidrug transporter protein.

Variants of a carbohydrate utilization-related or multidrug transporterprotein that function as either agonists or antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of a carbohydrate utilization-related or multidrugtransporter protein for agonist or antagonist activity. In oneembodiment, a variegated library of carbohydrate utilization-relatedvariants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof carbohydrate utilization-related or multidrug transporter variantscan be produced by, for example, enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences such that a degenerateset of potential carbohydrate utilization-related or multidrugtransporter sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of carbohydrate utilization-related ormultidrug transporter sequences therein. There are a variety of methodsthat can be used to produce libraries of potential carbohydrateutilization-related or multidrug transporter variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potentialcarbohydrate utilization-related or multidrug transporter sequences.Methods for synthesizing degenerate oligonucleotides are known in theart (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984)Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ikeet al. (1983) Nucleic Acids Res. 11:477).

In addition, libraries of fragments of a carbohydrateutilization-related or multidrug transporter protein coding sequence canbe used to generate a variegated population of carbohydrateutilization-related or multidrug transporter fragments for screening andsubsequent selection of variants of a carbohydrate utilization-relatedor multidrug transporter protein. In one embodiment, a library of codingsequence fragments can be generated by treating a double-stranded PCRfragment of a carbohydrate utilization-related or multidrug transportercoding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double-stranded DNA,renaturing the DNA to form double-stranded DNA which can includesense/antisense pairs from different nicked products, removingsingle-stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, one can derive an expression library thatencodes N-terminal and internal fragments of various sizes of thecarbohydrate utilization-related or multidrug transporter protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of carbohydrateutilization-related or multidrug transporter proteins. The most widelyused techniques, which are amenable to high through-put analysis, forscreening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquethat enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identifycarbohydrate utilization-related or multidrug transporter variants(Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Sequence Identity

The carbohydrate utilization-related and multidrug transporter sequencesare members of families of molecules with conserved functional features.By “family” is meant two or more proteins or nucleic acids havingsufficient nucleotide or amino acid sequence identity. By “sequenceidentity” is meant the nucleotide or amino acid residues that are thesame when aligning two sequences for maximum correspondence over aspecified comparison window. By “comparison window” is meant acontiguous segment of the two nucleotide or amino acid sequences foroptimal alignment, wherein the second sequence may contain additions ordeletions (i.e., gaps) as compared to the first sequence. Generally, fornucleic acid alignments, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.For amino acid sequence alignments, the comparison window is at least 6contiguous amino acids in length, and optionally can be 10, 15, 20, 30,or longer. Those of skill in the art understand that to avoid a highsimilarity due to inclusion of gaps, a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Family members may be from the same or different species, and caninclude homologues as well as distinct proteins. Often, members of afamily display common functional characteristics. Homologues can beisolated based on their identity to the Lactobacillus acidophiluscarbohydrate utilization-related or multidrug transporter nucleic acidsequences disclosed herein using the cDNA, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions as disclosed below.

To determine the percent identity of two amino acid or nucleotidesequences, an alignment is performed. Percent identity of the twosequences is a function of the number of identical residues shared bythe two sequences in the comparison window (i.e., percentidentity=number of identical residues/total number of residues×100). Inone embodiment, the sequences are the same length. Methods similar tothose mentioned below can be used to determine the percent identitybetween two sequences. The methods can be used with or without allowinggaps. Alignment may also be performed manually by inspection.

When amino acid sequences differ in conservative substitutions, thepercent identity may be adjusted upward to correct for the conservativenature of the substitution. Means for making this adjustment are knownin the art. Typically the conservative substitution is scored as apartial, rather than a full mismatch, thereby increasing the percentagesequence identity.

Mathematical algorithms can be used to determine the percent identity oftwo sequences. Non-limiting examples of mathematical algorithms are thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877; the algorithm of Myers and Miller (1988) CABIOS4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl.Math. 2:482; the global alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443-453; and the search-for-local-alignmentmethod of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA85:2444-2448.

Various computer implementations based on these mathematical algorithmshave been designed to enable the determination of sequence identity. TheBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are basedon the algorithm of Karlin and Altschul (1990) supra. Searches to obtainnucleotide sequences that are homologous to nucleotide sequences of thepresent invention can be performed with the BLASTN program, score=100,wordlength=12. To obtain amino acid sequences homologous to sequencesencoding a protein or polypeptide of the current invention, the BLASTXprogram may be used, score=50, wordlength=3. Gapped alignments may beobtained by using Gapped BLAST (in BLAST 2.0) as described in Altschulet al. (1997) Nucleic Acids Res. 25:3389. To detect distantrelationships between molecules, PSI-BLAST can be used. See, Altschul etal. (1997) supra. For all of the BLAST programs, the default parametersof the respective programs can be used. Alignment may also be performedmanually by inspection.

Another program that can be used to determine percent sequence identityis the ALIGN program (version 2.0), which uses the mathematicalalgorithm of Myers and Miller (1988) supra. A PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be usedwith this program when comparing amino acid sequences.

In addition to the ALIGN and BLAST programs, the BESTFIT, GAP, FASTA andTFASTA programs are part of the GCG Wisconsin Genetics Software Package,Version 10 (available from Accelrys Inc., 9685 Scranton Rd., San Diego,Calif., USA), and can be used for performing sequence alignments. Thepreferred program is GAP version 10, which used the algorithm ofNeedleman and Wunsch (1970) supra. Unless otherwise stated the sequenceidentity values provided herein refer to those values obtained by usingGAP Version 10 with the following parameters: % identity and %similarity for a nucleotide sequence using GAP Weight of 50 and LengthWeight of 3, and the nwsgapdna.cmp scoring matrix; % identity and %similarity for an amino acid sequence using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix; or any equivalent programthereof. By “equivalent program” is meant any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by GAP Version 10.

Alignment of a sequence in a database to a queried sequence produced byBLASTN, FASTA, BLASTP or like algorithm is commonly described as a“hit.” Hits to one or more database sequences by a queried sequenceproduced by BLASTN, FASTA, BLASTP or a similar algorithm, align andidentify similar portions of a sequence. A hit to a database sequencegenerally represents an overlap over a fraction of the sequence lengthof the queried sequence, i.e., a portion or fragment of the queriedsequence. However, the overlap can represent the entire length of thequeried sequence. The hits in an alignment to a queried sequenceproduced by BLASTN, FASTA, or BLASTP algorithms to sequences in adatabase are commonly arranged in order of the degree of similarity andthe length of sequence overlap.

Polynucleotide and polypeptide hits aligned by BLASTN, FASTA, or BLASTPalgorithms to a queried sequence produce “Expect” values. The Expectvalue (E value) indicates the number of hits one can “expect” to seeover a certain number of contiguous sequences at random when searching adatabase of a certain size. The Expect value is used as a significancethreshold for determining whether the hit to a database, such as theGenBank or the EMBL database, indicates actual similarity. For example,an E value of 0.1 assigned to a polynucleotide hit is interpreted asmeaning that in a database of the size of the GenBank database, onemight expect to see 0.1 matches over the aligned portion of the sequencewith a similar score randomly. By this criterion, the aligned andmatched portions of the polynucleotide sequences then have a probabilityof 90% of being the same. For sequences having an E value of 0.01 orless over aligned and matched portions, the probability of finding amatch randomly in the GenBank database is 1% or less, using the BLASTNor FASTA algorithm.

According to an embodiment of this invention, “variant” polynucleotidesand polypeptides of this invention, comprise sequences producing an Evalue of about 0.01 or less when compared to the polynucleotide orpolypeptide sequences of the present invention. That is, a variantpolynucleotide or polypeptide is any sequence that has at least a 99%probability of being the same as the polynucleotide or polypeptide ofthe present invention, measured as having an E value of 0.01 or lessusing the BLASTN, FASTA, or BLASTP algorithms set at parametersdescribed herein. In other embodiments, a variant polynucleotide is asequence having the same number of, or fewer nucleic acids than apolynucleotide of the present invention that has at least a 99%probability of being the same as the polynucleotide of the presentinvention, measured as having an E value of 0.01 or less using theBLASTN or FASTA algorithms set at parameters described herein.Similarly, a variant polypeptide is a sequence having the same numberof, or fewer amino acids than a polypeptide of the present inventionthat has at least a 99% probability of being the same as a polypeptideof the present invention, measured as having an E value of 0.01 or lessusing the BLASTP algorithm set at the parameters described herein.

As noted above, the percentage identity is determined by aligningsequences using one of the BLASTN, FASTA, or BLASTP algorithms, set atthe running parameters described herein, and identifying the number ofidentical nucleic acids or amino acids over the aligned portions;dividing the number of identical nucleic acids or amino acids by thetotal number of nucleic acids or amino acids of the polynucleotide orpolypeptide sequence of the present invention; and then multiplying by100 to determine the percent identity. For example, a polynucleotide ofthe present invention having 220 nucleic acids has a hit to apolynucleotide sequence in the GenBank database having 520 nucleic acidsover a stretch of 23 nucleotides in the alignment produced by the BLASTNalgorithm using the parameters described herein. The 23 nucleotide hitincludes 21 identical nucleotides, one gap and one different nucleotide.The percent identity of the polynucleotide of the present invention tothe hit in the GenBank library is thus 21/220 times 100, or 9.5%. Thepolynucleotide sequence in the GenBank database is thus not a variant ofa polynucleotide of the present invention.

Identification and Isolation of Homologous Sequences

Carbohydrate utilization-related nucleotide sequences identified basedon their sequence identity to the carbohydrate utilization-related ormultidrug transporter nucleotide sequences set forth herein or tofragments and variants thereof are encompassed by the present invention.Methods such as PCR or hybridization can be used to identify sequencesfrom a cDNA or genomic library, for example that are substantiallyidentical to a sequence of the invention. See, for example, Sambrook etal. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NewYork). Methods for construction of such cDNA and genomic libraries aregenerally known in the art and are also disclosed in the abovereference.

In hybridization techniques, the hybridization probes may be genomic DNAfragments, cDNA fragments, RNA fragments, or other oligonucleotides, andmay consist of all or part of a known nucleotide sequence disclosedherein. In addition, they may be labeled with a detectable group such as³²P, or any other detectable marker, such as other radioisotopes, afluorescent compound, an enzyme, or an enzyme co-factor. Probes forhybridization may be made by labeling synthetic oligonucleotides basedon the known carbohydrate utilization-related or multidrug transporternucleotide sequences disclosed herein. Degenerate primers designed onthe basis of conserved nucleotides or amino acid residues in a knowncarbohydrate utilization-related or multidrug transporter nucleotidesequence or encoded amino acid sequence can additionally be used. Thehybridization probe typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 10,preferably about 20, more preferably about 50, 75, 100, 125, 150, 175,200, 250, 300, 350, or 400 consecutive nucleotides of a nucleotidesequence of the invention or a fragment or variant thereof. To achievespecific hybridization under a variety of conditions, such probesinclude sequences that are unique among carbohydrate utilization-relatedor multidrug transporter protein sequences. Preparation of probes forhybridization is generally known in the art and is disclosed in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., ColdSpring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated byreference.

In one embodiment, the entire nucleotide sequence encoding acarbohydrate utilization-related or multidrug transporter protein isused as a probe to identify novel carbohydrate utilization-related ormultidrug transporter sequences and messenger RNAs. In anotherembodiment, the probe is a fragment of a nucleotide sequence disclosedherein. In some embodiments, the nucleotide sequence that hybridizesunder stringent conditions to the probe can be at least about 300, 325,350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200,1400, 1600, 1800, or 2000 nucleotides in length.

Substantially identical sequences will hybridize to each other understringent conditions. By “stringent conditions” is meant conditionsunder which a probe will hybridize to its target sequence to adetectably greater degree than to other sequences (e.g., at least 2-foldover background). Generally, stringent conditions encompass thoseconditions for hybridization and washing under which nucleotides havingat least about 60%, 65%, 70%, preferably 75% sequence identity typicallyremain hybridized to each other. Stringent conditions are known in theart and can be found in Current Protocols in Molecular Biology (JohnWiley & Sons, New York (1989)), 6.3.1-6.3.6. Hybridization typicallyoccurs for less than about 24 hours, usually about 4 to about 12 hours.

Stringent conditions are sequence dependent and will differ in differentcircumstances. Full-length or partial nucleic acid sequences may be usedto obtain homologues and orthologs encompassed by the present invention.By “orthologs” is meant genes derived from a common ancestral gene andwhich are found in different species as a result of speciation. Genesfound in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences sharesubstantial identity as defined elsewhere herein. Functions of orthologsare often highly conserved among species.

When using probes, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides).

The post-hybridization washes are instrumental in controllingspecificity. The two critical factors are ionic strength and temperatureof the final wash solution. For the detection of sequences thathybridize to a full-length or approximately full-length target sequence,the temperature under stringent conditions is selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. However, stringent conditions wouldencompass temperatures in the range of 1° C. to 20° C. lower than theT_(m), depending on the desired degree of stringency as otherwisequalified herein. For DNA-DNA hybrids, the T_(m) can be determined usingthe equation of Meinkoth and Wahl (1984) Anal, Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (logM)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe.

The ability to detect sequences with varying degrees of homology can beobtained by varying the stringency of the hybridization and/or washingconditions. To target sequences that are 100% identical (homologousprobing), stringency conditions must be obtained that do not allowmismatching. By allowing mismatching of nucleotide residues to occur,sequences with a lower degree of similarity can be detected(heterologous probing). For every 1% of mismatching, the T_(m) isreduced about 1° C.; therefore, hybridization and/or wash conditions canbe manipulated to allow hybridization of sequences of a targetpercentage identity. For example, if sequences with ≧90% sequenceidentity are preferred, the T_(m) can be decreased by 10° C. Twonucleotide sequences could be substantially identical, but fail tohybridize to each other under stringent conditions, if the polypeptidesthey encode are substantially identical. This situation could arise, forexample, if the maximum codon degeneracy of the genetic code is used tocreate a copy of a nucleic acid.

Exemplary low stringency conditions include hybridization with a buffersolution of 30-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate)at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50 to 55° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, washbuffers may comprise about 0.1% to about 1% SDS. Duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours. An extensive guide to the hybridization of nucleic acidsis found in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed.; Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. PCR primers arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Methods for designing PCRprimers and PCR cloning are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See alsoInnis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

Assays

Diagnostic assays to detect expression of the disclosed polypeptidesand/or nucleic acids as well as their disclosed activity in a sample aredisclosed. An exemplary method for detecting the presence or absence ofa disclosed nucleic acid or protein comprising the disclosed polypeptidein a sample involves obtaining a sample from a food/dairy/feed product,starter culture (mother, seed, bulk/set, concentrated, dried,lyophilized, frozen), cultured food/dairy/feed product, dietarysupplement, bioprocessing fermentate, or a subject that has ingested aprobiotic material, and contacting the sample with a compound or anagent capable of detecting the disclosed polypeptides or nucleic acids(e.g., an mRNA or genomic DNA comprising the disclosed nucleic acid orfragment thereof) such that the presence of the disclosed sequence isdetected in the sample. Results obtained with a sample from the food,supplement, culture, product, or subject may be compared to resultsobtained with a sample from a control culture, product, or subject.

One agent for detecting the mRNA or genomic DNA comprising a disclosednucleotide sequence is a labeled nucleic acid probe capable ofhybridizing to the disclosed nucleotide sequence of the mRNA or genomicDNA. The nucleic acid probe can be, for example, a disclosed nucleicacid, such as a nucleic acid of odd numbered SEQ ID NOS:1-321, or aportion thereof, such as a nucleic acid of at least 15, 30, 50, 100,250, or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to the mRNA or genomic DNAcomprising the disclosed nucleic acid sequence. Other suitable probesfor use in the diagnostic assays of the invention are described herein.

One agent for detecting a protein comprising a disclosed polypeptidesequence is an antibody capable of binding to the disclosed polypeptide,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(abN)₂) can be used. The term “labeled,”with regard to the probe or antibody, is meant to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin.

The term “sample” is meant to include tissues, cells, and biologicalfluids present in or isolated from a subject, as well as cells fromstarter cultures or food products carrying such cultures, or derivedfrom the use of such cultures. That is, the detection method of theinvention can be used to detect mRNA, protein, or genomic DNA comprisinga disclosed sequence in a sample both in vitro and in vivo. In vitrotechniques for detection of mRNA comprising a disclosed sequence includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of a protein comprising a disclosed polypeptide includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of genomic DNA comprising the disclosed nucleotide sequencesinclude Southern hybridizations. Furthermore, in vivo techniques fordetection of a protein comprising a disclosed polypeptide includeintroducing into a subject a labeled antibody against the disclosedpolypeptide. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

In one embodiment, the sample contains protein molecules from a testsubject that has consumed a probiotic material. Alternatively, thesample can contain mRNA or genomic DNA from a starter culture.

The invention also encompasses kits for detecting the presence ofdisclosed nucleic acids or proteins comprising disclosed polypeptides ina sample. Such kits can be used to determine if a microbe expressing aspecific polypeptide of the invention is present in a food product orstarter culture, or in a subject that has consumed a probiotic material.For example, the kit can comprise a labeled compound or agent capable ofdetecting a disclosed polypeptide or mRNA in a sample and means fordetermining the amount of a the disclosed polypeptide in the sample(e.g., an antibody that recognizes the disclosed polypeptide or anoligonucleotide probe that binds to DNA encoding a disclosedpolypeptide, e.g., even numbered SEQ ID NOS:2-322). Kits can alsoinclude instructions detailing the use of such compounds.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to a disclosedpolypeptide; and, optionally, (2) a second, different antibody thatbinds to the disclosed polypeptide or the first antibody and isconjugated to a detectable agent. For oligonucleotide-based kits, thekit can comprise, for example: (1) an oligonucleotide, e.g., adetectably labeled oligonucleotide, that hybridizes to a disclosednucleic acid sequence or (2) a pair of primers useful for amplifying adisclosed nucleic acid.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for use.

In one embodiment, the kit comprises multiple probes in an array format,such as those described, for example, in U.S. Pat. Nos. 5,412,087 and5,545,531, and International Publication No. WO 95/00530, hereinincorporated by reference. Probes for use in the array may besynthesized either directly onto the surface of the array, as disclosedin International Publication No. WO 95/00530, or prior to immobilizationonto the array surface (Gait, ed. (1984) Oligonucleotide Synthesis aPractical Approach IRL Press, Oxford, England). The probes may beimmobilized onto the surface using techniques well known to one of skillin the art, such as those described in U.S. Pat. No. 5,412,087. Probesmay be a nucleic acid or peptide sequence, preferably purified, or anantibody.

The arrays may be used to screen organisms, samples, or products fordifferences in their genomic, cDNA, polypeptide, or antibody content,including the presence or absence of specific sequences or proteins, aswell as the concentration of those materials. Binding to a capture probeis detected, for example, by signal generated from a label attached tothe nucleic acid comprising the disclosed nucleic acid sequence, apolypeptide comprising the disclosed amino acid sequence, or anantibody. The method can include contacting the molecule comprising thedisclosed nucleic acid, polypeptide, or antibody with a first arrayhaving a plurality of capture probes and a second array having adifferent plurality of capture probes. The results of each hybridizationcan be compared to analyze differences in expression between a first andsecond sample. The first plurality of capture probes can be from acontrol sample, e.g., a wild type lactic acid bacteria, or controlsubject, e.g., a food, dietary supplement, starter culture sample, or abiological fluid. The second plurality of capture probes can be from anexperimental sample, e.g., a mutant type lactic acid bacteria, orsubject that has consumed a probiotic material, e.g., a starter culturesample or a biological fluid.

These assays may be especially useful in microbial selection and qualitycontrol procedures where the detection of unwanted materials isessential. The detection of particular nucleotide sequences orpolypeptides may also be useful in determining the genetic compositionof food, fermentation products, or industrial microbes, or microbespresent in the digestive system of animals or humans that have consumedprobiotics.

Antisense Nucleotide Sequences

The present invention also encompasses antisense nucleic acids, i.e.,molecules that are complementary to a sense nucleic acid encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire carbohydrateutilization-related or multidrug transporter coding strand, or to only aportion thereof, e.g., all or part of the protein coding region (or openreading frame). An antisense nucleic acid can be antisense to anoncoding region of the coding strand of a nucleotide sequence encodinga carbohydrate utilization-related or multidrug transporter protein. Thenoncoding regions are the 5′ and 3′ sequences that flank the codingregion and are not translated into amino acids. Antisense nucleotidesequences are useful in disrupting the expression of the target gene.Antisense constructions having 70%, preferably 80%, more preferably 85%,90% or 95% sequence identity to the corresponding sequence may be used.

Given the coding-strand sequence encoding a carbohydrateutilization-related or multidrug transporter protein disclosed herein(e.g., even numbered SEQ ID NOS:2-322), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid can be complementary to theentire coding region of carbohydrate utilization-related or multidrugtransporter mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region ofcarbohydrate utilization-related or multidrug transporter mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of carbohydrateutilization-related or multidrug transporter mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45, or 50 nucleotides in length, or it can be 100, 200 nucleotides,or greater in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation proceduresknown in the art.

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest).

An antisense nucleic acid of the invention can be an α-anomeric nucleicacid. An α-anomeric nucleic acid forms specific double-stranded hybridswith complementary RNA in which, contrary to the usual β-units, thestrands run parallel to each other (Gaultier et al. (1987) Nucleic AcidsRes. 15:6625-6641). The antisense nucleic acid can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave carbohydrate utilization-related mRNA transcriptsto thereby inhibit translation of carbohydrate utilization-related ormultidrug transporter mRNA. A ribozyme having specificity for ancarbohydrate utilization-related-encoding or multidrugtransporter-encoding nucleic acid can be designed based upon thenucleotide sequence of an carbohydrate utilization-related or multidrugtransporter cDNA disclosed herein (e.g., odd numbered SEQ ID NOS:1-320).See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S.Pat. No. 5,116,742. Alternatively, carbohydrate utilization-related ormultidrug transporter mRNA can be used to select a catalytic RNA havinga specific ribonuclease activity from a pool of RNA molecules. See,e.g., Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acids that form triple helicalstructures. For example, carbohydrate utilization-related or multidrugtransporter gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of the carbohydrateutilization-related or multidrug transporter protein (e.g., thecarbohydrate utilization-related or multidrug transporter promoterand/or enhancers) to form triple helical structures that preventtranscription of the carbohydrate utilization-related or multidrugtransporter gene in target cells. See generally, Helene (1991)Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N.Y. Acad. Sci.660:27; and Maher (1992) Bioassays 14(12):807.

In some embodiments, the nucleic acids of the invention can be modifiedat the base moiety, sugar moiety, or phosphate backbone to improve,e.g., the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of the nucleic acids can bemodified to generate peptide nucleic acids (see Hyrup et al. (1996)Bioorganic & Medicinal Chemistry 4:5). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid-phase peptide synthesis protocols as described, for example, inHyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad.Sci. USA 93:14670.

PNAs can be used as antisense or antigene agents for sequence-specificmodulation of gene expression by, e.g., inducing transcription ortranslation arrest or inhibiting replication. PNAs of the invention canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA-directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996) supra); or as probes or primers for DNA sequence andhybridization (Hyrup (1996) supra; Perry-O'Keefe et al. (1996) supra).

In another embodiment, PNAs of an carbohydrate utilization-related ormultidrug transporter molecule can be modified, e.g., to enhance theirstability, specificity, or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. The synthesis of PNA-DNA chimeras can be performed as described inHyrup (1996) supra; Finn et al. (1996) Nucleic Acids Res.24(17):3357-63; Mag et al. (1989) Nucleic Acids Res. 17:5973; andPeterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

Fusion Proteins

The invention also includes carbohydrate utilization-related ormultidrug transporter chimeric or fusion proteins. A carbohydrateutilization-related or multidrug transporter “chimeric protein” or“fusion protein” comprises a carbohydrate utilization-related ormultidrug transporter polypeptide operably linked to a non-carbohydrateutilization-related or non-multidrug transporter polypeptide,respectively. A “carbohydrate utilization-related polypeptide” or a“multidrug transporter polypeptide” refers to a polypeptide having anamino acid sequence corresponding to a carbohydrate utilization-relatedprotein or a multidrug transporter protein, respectively, whereas a“non-carbohydrate utilization-related polypeptide” or a “non-multidrugtransporter polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein that is not substantially identicalto the carbohydrate utilization-related protein or multidrug transporterprotein, respectively, and which is derived from the same or a differentorganism. Within a carbohydrate utilization-related or multidrugtransporter fusion protein, the carbohydrate utilization-related ormultidrug transporter polypeptide can correspond to all or a portion ofa carbohydrate utilization-related or multidrug transporter protein,preferably including at least one biologically active portion of acarbohydrate utilization-related or multidrug transporter protein.Within the fusion protein, the term “operably linked” is meant toindicate that the carbohydrate utilization-related or multidrugtransporter polypeptide and the non-carbohydrate utilization-related ormultidrug transporter polypeptide are fused in-frame to each other. Thenon-carbohydrate utilization-related or multidrug transporterpolypeptide can be fused to the N-terminus or C-terminus of thecarbohydrate utilization-related or multidrug transporter polypeptide.

Expression of the linked coding sequences results in two linkedheterologous amino acid sequences that form the fusion protein. Thecarrier sequence (the non-carbohydrate utilization-related ornon-multidrug transporter polypeptide) can encode a carrier polypeptidethat potentiates or increases expression of the fusion protein in thebacterial host. The portion of the fusion protein encoded by the carriersequence, i.e., the carrier polypeptide, may be a protein fragment, anentire functional moiety, or an entire protein sequence. The carrierregion or polypeptide may additionally be designed to be used inpurifying the fusion protein, either with antibodies or with affinitypurification specific for that carrier polypeptide. Likewise, physicalproperties of the carrier polypeptide can be exploited to allowselective purification of the fusion protein.

Particular carrier polypeptides of interest include superoxide dismutase(SOD), maltose-binding protein (MBP), glutathione-S-transferase (GST),an N-terminal histidine (His) tag, and the like. This list is not meantto be limiting, as any carrier polypeptide that potentiates expressionof the carbohydrate utilization-related protein or multidrug resistanceprotein as a fusion protein can be used in the methods of the invention.

In one embodiment, the fusion protein is a GST-carbohydrateutilization-related fusion protein in which the carbohydrateutilization-related sequences are fused to the C-terminus of the GSTsequences. In another embodiment, the fusion protein is a carbohydrateutilization-related-immunoglobulin fusion protein in which all or partof a carbohydrate utilization-related protein is fused to sequencesderived from a member of the immunoglobulin protein family. In otherembodiments, the fusion protein comprises a multidrug transporterprotein of the present invention. The carbohydrate utilization-related-or multidrug transporter-immunoglobulin fusion proteins of the inventioncan be used as immunogens to produce anti-carbohydrateutilization-related or anti-multidrug transporter-related antibodies ina subject, to purify carbohydrate utilization-related or multidrugtransporter-related ligands, and in screening assays to identifymolecules that inhibit the interaction of a carbohydrateutilization-related or multidrug transporter protein with a carbohydrateutilization-related or multidrug transporter ligand.

One of skill in the art will recognize that the particular carrierpolypeptide is chosen with the purification scheme in mind. For example,His tags, GST, and maltose-binding protein represent carrierpolypeptides that have readily available affinity columns to which theycan be bound and eluted. Thus, where the carrier polypeptide is anN-terminal His tag such as hexahistidine (His₆ tag), the carbohydrateutilization-related or multidrug transporter fusion protein can bepurified using a matrix comprising a metal-chelating resin, for example,nickel nitrilotriacetic acid (Ni-NTA), nickel iminodiacetic acid(Ni-IDA), and cobalt-containing resin (Co-resin). See, for example,Steinert et al. (1997) QIAGEN News 4:11-15, herein incorporated byreference in its entirety. Where the carrier polypeptide is GST, thecarbohydrate utilization-related or multidrug transporter fusion proteincan be purified using a matrix comprising glutathione-agarose beads(Sigma or Pharmacia Biotech); where the carrier polypeptide is amaltose-binding protein (MBP), the carbohydrate utilization-related ormultidrug transporter fusion protein can be purified using a matrixcomprising an agarose resin derivatized with amylose.

Preferably, a chimeric or fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences may be ligated togetherin-frame, or the fusion gene can be synthesized, such as with automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments, which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology(Greene Publishing and Wiley-Interscience, New York). Moreover, acarbohydrate utilization-related or multidrug transporter-encodingnucleic acid can be cloned into a commercially available expressionvector such that it is linked in-frame to an existing fusion moiety.

The fusion protein expression vector is typically designed for ease ofremoving the carrier polypeptide to allow the carbohydrateutilization-related or multidrug transporter protein to retain thenative biological activity associated with it. Methods for cleavage offusion proteins are known in the art. See, for example, Ausubel et al.,eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons,Inc.). Chemical cleavage of the fusion protein can be accomplished withreagents such as cyanogen bromide,2-(2-nitrophenylsulphenyl)-3-methyl-3′-bromoindolenine, hydroxylamine,or low pH. Chemical cleavage is often accomplished under denaturingconditions to cleave otherwise insoluble fusion proteins.

Where separation of the carbohydrate utilization-related or multidrugtransporter polypeptide from the carrier polypeptide is desired and acleavage site at the junction between these fused polypeptides is notnaturally occurring, the fusion construct can be designed to contain aspecific protease cleavage site to facilitate enzymatic cleavage andremoval of the carrier polypeptide. In this manner, a linker sequencecomprising a coding sequence for a peptide that has a cleavage sitespecific for an enzyme of interest can be fused in-frame between thecoding sequence for the carrier polypeptide (for example, MBP, GST, SOD,or an N-terminal His tag) and the coding sequence for the carbohydrateutilization-related or multidrug transporter polypeptide. Suitableenzymes having specificity for cleavage sites include, but are notlimited to, factor Xa, thrombin, enterokinase, remin, collagenase, andtobacco etch virus (TEV) protease. Cleavage sites for these enzymes arewell known in the art. Thus, for example, where factor Xa is to be usedto cleave the carrier polypeptide from the carbohydrateutilization-related or multidrug transporter polypeptide, the fusionconstruct can be designed to comprise a linker sequence encoding afactor Xa-sensitive cleavage site, for example, the sequence IEGR (see,for example, Nagai and Thøgersen (1984) Nature 309:810-812, Nagai andThøgersen (1987) Meth. Enzymol. 153:461-481, and Pryor and Leiting(1997) Protein Expr. Purif: 10(3):309-319, herein incorporated byreference). Where thrombin is to be used to cleave the carrierpolypeptide from the carbohydrate utilization-related or multidrugtransporter polypeptide, the fusion construct can be designed tocomprise a linker sequence encoding a thrombin-sensitive cleavage site,for example the sequence LVPRGS or VIAGR (see, for example, Pryor andLeiting (1997) Protein Expr. Purif 10(3):309-319, and Hong et al. (1997)Chin. Med. Sci. J. 12(3):143-147, respectively, herein incorporated byreference). Cleavage sites for TEV protease are known in the art. See,for example, the cleavage sites described in U.S. Pat. No. 5,532,142,herein incorporated by reference in its entirety. See also thediscussion in Ausubel et al., eds. (1998) Current Protocols in MolecularBiology (John Wiley & Sons, Inc.), Chapter 16.

Antibodies

An isolated polypeptide of the present invention can be used as animmunogen to generate antibodies that specifically bind carbohydrateutilization-related or multidrug transporter proteins, or stimulateproduction of antibodies in vivo. The full-length carbohydrateutilization-related or multidrug transporter protein can be used as animmunogen or, alternatively, antigenic peptide fragments of carbohydrateutilization-related or multidrug transporter proteins as describedherein can be used. The antigenic peptide of an carbohydrateutilization-related or multidrug transporter protein comprises at least8, preferably 10, 15, 20, or 30 amino acid residues of the amino acidsequences shown in even numbered SEQ ID NOS:1-320 and encompasses anepitope of a carbohydrate utilization-related or multidrug transporterprotein such that an antibody raised against the peptide forms aspecific immune complex with the carbohydrate utilization-related ormultidrug transporter protein. Preferred epitopes encompassed by theantigenic peptide are regions of a carbohydrate utilization-related ormultidrug transporter protein that are located on the surface of theprotein, e.g., hydrophilic regions.

Recombinant Expression Vectors and Cells

The nucleic acids of the present invention may be included in vectors,preferably expression vectors. “Vector” refers to a nucleic acid capableof transporting another nucleic acid to which it has been linked.Expression vectors include one or more regulatory sequences and directthe expression of genes to which they are operably linked. By “operablylinked” is meant that the nucleotide sequence of interest is linked tothe regulatory sequence(s) such that expression of the nucleotidesequence is allowed (e.g., in an in vitro transcription/translationsystem or in a cell when the vector is introduced into the cell). Theterm “regulatory sequence” is meant to include controllabletranscriptional promoters, operators, enhancers, transcriptionalterminators, and other expression control elements such as translationalcontrol sequences (e.g., Shine-Dalgarno consensus sequence, initiationand termination codons). These regulatory sequences will differ, forexample, depending on the cell being used.

The vectors can be autonomously replicated in a cell (episomal vectors),or may be integrated into the genome of a cell, and replicated alongwith the host genome (non-episomal mammalian vectors). Integratingvectors typically contain at least one sequence homologous to thebacterial chromosome that allows for recombination to occur betweenhomologous DNA in the vector and the bacterial chromosome. Integratingvectors may also comprise bacteriophage or transposon sequences.Episomal vectors, or plasmids are circular double-stranded DNA loopsinto which additional DNA segments can be ligated. Plasmids capable ofstable maintenance in a host are generally the preferred form ofexpression vectors when using recombinant DNA techniques.

The expression constructs or vectors encompassed in the presentinvention comprise a nucleic acid construct of the invention in a formsuitable for expression of the nucleic acid in a cell. Expression inprokaryotic cells and plant cells is encompassed in the presentinvention. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto cells to thereby produce proteins or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein(e.g., carbohydrate utilization-related or multidrug transporterproteins, mutant forms of carbohydrate utilization-related or multidrugtransporter proteins, fusion proteins, etc.).

Bacterial Expression Vectors

Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence as well as those that direct inducibleexpression of the nucleotide sequence only under certain environmentalconditions. A bacterial promoter is any DNA sequence capable of bindingbacterial RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region, which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A bacterial promoter may also have asecond domain called an operator, which may overlap an adjacent RNApolymerase binding site at which RNA synthesis begins. The operatorpermits negative regulated (inducible) transcription, as a generepressor protein may bind the operator and thereby inhibittranscription of a specific gene. Constitutive expression may occur inthe absence of negative regulatory elements, such as the operator. Inaddition, positive regulation may be achieved by a gene activatorprotein binding sequence, which, if present is usually proximal (5′) tothe RNA polymerase binding sequence.

An example of a gene activator protein is the catabolite activatorprotein (CAP), which helps initiate transcription of the lac operon inEscherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173).Regulated expression may therefore be either positive or negative,thereby either enhancing or reducing transcription. Other examples ofpositive and negative regulatory elements are well known in the art.Various promoters that can be included in the protein expression systeminclude, but are not limited to, a T7/LacO hybrid promoter, a trppromoter, a T7 promoter, a lac promoter, and a bacteriophage lambdapromoter. Any suitable promoter can be used to carry out the presentinvention, including the native promoter or a heterologous promoter.Heterologous promoters may be constitutively active or inducible. Anon-limiting example of a heterologous promoter is given in U.S. Pat.No. 6,242,194.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) (Chang etal. (1987) Nature 198:1056), and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; Yelverton et al.(1981) Nucleic Acids Res. 9:731; U.S. Pat. No. 4,738,921; EPOPublication Nos. 36,776 and 121,775). The beta-lactamase (bla) promotersystem (Weissmann, (1981) “The Cloning of Interferon and OtherMistakes,” in Interferon 3 (ed. I. Gresser); bacteriophage lambda PL(Shimatake et al. (1981) Nature 292:128); the arabinose-inducible araBpromoter (U.S. Pat. No. 5,028,530); and T5 (U.S. Pat. No. 4,689,406)promoter systems also provide useful promoter sequences. See also Balbas(2001) Mol. Biotech. 19:251-267, where E. coli expression systems arediscussed.

In addition, synthetic promoters that do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter (U.S. Pat. No. 4,551,433). Forexample, the tac (Amann et al. (1983) Gene 25:167; de Boer et al. (1983)Proc. Natl. Acad. Sci. 80:21) and trc (Brosius et al. (1985) J. Biol.Chem. 260:3539-3541) promoters are hybrid trp-lac promoters comprised ofboth trp promoter and lac operon sequences that are regulated by the lacrepressor. The tac promoter has the additional feature of being aninducible regulatory sequence. Thus, for example, expression of a codingsequence operably linked to the tac promoter can be induced in a cellculture by adding isopropyl-1-thio-β-D-galactoside (IPTG). Furthermore,a bacterial promoter can include naturally occurring promoters ofnon-bacterial origin that have the ability to bind bacterial RNApolymerase and initiate transcription. A naturally occurring promoter ofnon-bacterial origin can also be coupled with a compatible RNApolymerase to produce high levels of expression of some genes inprokaryotes. The bacteriophage T7 RNA polymerase/promoter system is anexample of a coupled promoter system (Studier et al. (1986) J. Mol.Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074). Inaddition, a hybrid promoter can also be comprised of a bacteriophagepromoter and an E. coli operator region (EPO Publication No. 267,851).

The vector may additionally contain a gene encoding the repressor (orinducer) for that promoter. For example, an inducible vector of thepresent invention may regulate transcription from the Lac operator(LacO) by expressing the gene encoding the Lad repressor protein. Otherexamples include the use of the lexA gene to regulate expression ofpRecA, and the use of trpO to regulate ptrp. Alleles of such genes thatincrease the extent of repression (e.g., lacIq) or that modify themanner of induction (e.g., lambda CI857, rendering lambda pLthermo-inducible, or lambda CI+, rendering lambda pL chemo-inducible)may be employed.

In addition to a functioning promoter sequence, an efficientribosome-binding site is also useful for the expression of the fusionconstruct. In prokaryotes, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon (Shine et al. (1975) Nature 254:34). The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ end of bacterial 16SrRNA (Steitz et al. (1979) “Genetic Signals and Nucleotide Sequences inMessenger RNA,” in Biological Regulation and Development: GeneExpression (ed. R. F. Goldberger, Plenum Press, NY).

Carbohydrate utilization-related proteins can also be secreted from thecell by creating chimeric DNA molecules that encode a protein comprisinga signal peptide sequence fragment that provides for secretion of thecarbohydrate utilization-related and multidrug transporter polypeptidesin bacteria (U.S. Pat. No. 4,336,336). The signal sequence fragmenttypically encodes a signal peptide comprised of hydrophobic amino acidsthat direct the secretion of the protein from the cell. The protein iseither secreted into the growth media (Gram-positive bacteria) or intothe periplasmic space, located between the inner and outer membrane ofthe cell (Gram-negative bacteria). Preferably there are processingsites, which can be cleaved either in vivo or in vitro, encoded betweenthe signal peptide fragment and the carbohydrate utilization-related ormultidrug transporter protein.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb etal. (1984) EMBO J. 3:2437-2442) and the E. coli alkaline phosphatasesignal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci.82:7212). Other prokaryotic signals include, for example, the signalsequence from penicillinase, Ipp, or heat stable enterotoxin II leaders.

Typically, transcription termination sequences recognized by bacteriaare regulatory regions located 3′ to the translation stop codon andthus, together with the promoter, flank the coding sequence. Thesesequences direct the transcription of an mRNA that can be translatedinto the polypeptide encoded by the DNA. Transcription terminationsequences frequently include DNA sequences (of about 50 nucleotides)that are capable of forming stem loop structures that aid in terminatingtranscription. Examples include transcription termination sequencesderived from genes with strong promoters, such as the trp gene in E.coli as well as other biosynthetic genes.

The expression vectors will have a plurality of restriction sites forinsertion of the carbohydrate utilization-related or multidrugtransporter sequence so that it is under transcriptional regulation ofthe regulatory regions. Selectable marker genes that ensure maintenanceof the vector in the cell can also be included in the expression vector.Preferred selectable markers include those that confer resistance todrugs such as ampicillin, chloramphenicol, erythromycin, kanamycin(neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol.32:469). Selectable markers may also allow a cell to grow on minimalmedium, or in the presence of toxic metabolite and may includebiosynthetic genes, such as those in the histidine, tryptophan, andleucine biosynthetic pathways.

The regulatory regions may be native (homologous), or may be foreign(heterologous) to the cell and/or the nucleotide sequence of theinvention. The regulatory regions may also be natural or synthetic.Where the region is “foreign” or “heterologous” to the cell, it is meantthat the region is not found in the native cell into which the region isintroduced. Where the region is “foreign” or “heterologous” to thecarbohydrate utilization-related or multidrug transporter nucleotidesequence of the invention, it is meant that the region is not the nativeor naturally occurring region for the operably linked carbohydrateutilization-related or multidrug transporter nucleotide sequence of theinvention. For example, the region may be derived from phage. While itmay be preferable to express the sequences using heterologous regulatoryregions, native regions may be used. Such constructs would be expectedin some cases to alter expression levels of carbohydrateutilization-related or multidrug transporter proteins in the cell. Thus,the phenotype of the cell could be altered.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to carbohydrate utilization-related or multidrugtransporter mRNA. Regulatory sequences operably linked to a nucleic acidcloned in the antisense orientation can be chosen to direct thecontinuous or inducible expression of the antisense RNA molecule. Theantisense expression vector can be in the form of a recombinant plasmidor phagemid in which antisense nucleic acids are produced under thecontrol of a high efficiency regulatory region, the activity of whichcan be determined by the cell type into which the vector is introduced.For a discussion of the regulation of gene expression using antisensegenes see Weintraub et al. (1986) Reviews—Trends in Genetics, Vol. 1(1).

Alternatively, some of the above-described components can be puttogether in transformation vectors. Transformation vectors are typicallycomprised of a selectable market that is either maintained in a repliconor developed into an integrating vector, as described above.

Plant Expression Vectors

For expression in plant cells, the expression cassettes will comprise atranscriptional initiation region operably linked to a nucleotidesequence of the present invention. Various restriction sites may beincluded in these expression vectors to enable insertion of thenucleotide sequence under the transcriptional regulation of theregulatory regions. Additionally, the expression cassette may containselectable marker genes, including those genes that provide herbicide orantibiotic resistance, such as tetracycline resistance, hygromycinresistance, ampicillin resistance, or glyphosate resistance.

The expression cassette will include in the 5′-to-3′ direction oftranscription, a transcriptional and translational initiation region, anucleotide sequence of the invention, and a transcriptional andtranslational termination region (i.e., termination region) functionalin plants. The termination region may be native with the transcriptionalinitiation region comprising the promoter nucleotide sequence, may benative with the nucleotide sequence of the invention, or may be derivedfrom another source. Convenient termination regions are known in the artand include, but are not limited to, a termination region from theTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also, Guerineau et al. (1991) Mol.Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon etal. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. 1989)Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic AcidRes. 15:9627-9639.

The expression cassette comprising a nucleotide sequence of the presentinvention may also contain at least one additional nucleotide sequencefor a gene to be cotransformed into the organism. Alternatively, theadditional sequence(s) may be provided on another expression cassette.

The expression cassettes may additionally contain 5′ non-translatedleader sequences or 5′ non-coding sequences. As used herein, “5′ leadersequence,” “translation leader sequence,” or “5′ non-coding sequence”refer to that DNA sequence portion of a gene between the promoter andcoding sequence that is transcribed into RNA and is present in the fullyprocessed mRNA upstream (5′) of the translation start codon. A 5′non-translated leader sequence is usually characterized as that portionof the mRNA molecule that most typically extends from the 5′ CAP site tothe AUG protein translation initiation codon. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency (Turner et al. (1995) MolecularBiotechnology 3:225). Thus, translation leader sequences play animportant role in the regulation of gene expression. Translation leadersare known in the art and include but are not limited to: picornavirusleaders, for example, EMCV leader (Encephalomyocarditis 5′ noncodingregion) (Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco EtchVirus) (Allison et al. (1986) Virology 154:9-20); MDMV leader (MaizeDwarf Mosaic Virus); human immunoglobulin heavy-chain binding protein(BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader fromthe coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling etal. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV)(Gallie et al. (1989) Molecular Biology of RNA, pages 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385).

Other methods known to enhance translation and/or mRNA stability canalso be utilized, for example, introns, such as the maize ubiquitinintron (Christensen and Quail (1996) Transgenic Res. 5:213-218 andChristensen et al. (1992) Plant Molecular Biology 18:675-689) or themaize AdhI intron (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48 andKyozuka et al. (1990) Maydica 35:353-357), and the like. Various intronsequences have been shown to enhance expression, particularly inmonocotyledonous cells. The introns of the maize AdhI gene have beenfound to significantly enhance the expression of the wild-type geneunder its cognate promoter when introduced into maize cells. Intron 1was found to be particularly effective and enhanced expression in fusionconstructs with the chloramphenicol acetyltransferase gene (Canis ei al.(1987) Genes Develop. 1:1183-1200). In the same experimental system, theintron from the maize bronzel gene had a similar effect in enhancingexpression. The AdhI intron has also been shown to enhance CATexpression 12-fold (Mascarenhas et al. (1990) Plant Mol. Biol.

6:913-920). Intron sequences have routinely been incorporated into planttransformation vectors, typically within the non-translated leader.

The expression cassette comprising a promoter sequence of the presentinvention may additionally contain a 3′ non-coding sequence. A “3′non-coding sequence” or “3′ non-translated region” refers to anucleotide sequence located 3′ (downstream) to a coding sequence andincludes polyadenylation signal sequences and other sequences encodingregulatory signals capable of affecting the addition of polyadenylicacid tracts to the 3′ end of the mRNA precursor. A 3′ non-translatedregion comprises a region of the mRNA generally beginning with thetranslation termination codon and extending at least beyond thepolyadenylation site. Non-translated sequences located in the 3′ end ofa gene have been found to influence gene expression levels. Ingelbrechtet al. (see, Plant Cell, 1:671-680, 1989) evaluated the importance ofthese elements and found large differences in expression in stableplants depending on the source of the 3′ non-translated region. Using 3′non-translated regions associated with octopine synthase, 2S seedprotein from Arabidopsis, small subunit of rbcS from Arabidopsis,extension from carrot, and chalcone synthase from Antirrhinium, a60-fold difference was observed between the best-expressing construct(which contained the rbcS 3′ non-translated region) and thelowest-expressing construct (which contained the chalcone synthase 3′region).

Transcription levels may also be increased by the utilization ofenhancers in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Adaptersor linkers may be employed to join the DNA fragments or othermanipulations may be involved to provide for convenient restrictionsites. Restriction sites may be added or removed, superfluous DNA may beremoved, or other modifications may be made to the sequences of theinvention. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, for example, transitions andtransversions, may be involved.

In addition to selectable markers that provide resistance to antibioticsor herbicides, as described above, other genes that could serve utilityin the recovery of transgenic events but might not be required in thefinal product would include, but are not limited to, GUS(b-glucoronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5:387), GFP(green florescence protein; Chalfie et al. (1994) Science 263:802),luciferase (Riggs et al. (1987) Nucleic Acids Res. 15(19):8115 andLuehrsen et al. (1992) Methods Enzymol. 216:397-114), and the maizegenes encoding for anthocyanin production (Ludwig et al. (1990) Science247:449).

The nucleic acids of the present invention are useful in methodsdirected to expressing a nucleotide sequence in a plant. This may beaccomplished by transforming a plant cell of interest with an expressioncassette comprising a promoter operably linked to a nucleotide sequenceidentified herein, and regenerating a stably transformed plant from saidplant cell. The expression cassette comprising the promoter sequenceoperably linked to a nucleotide sequence of the present invention can beused to transform any plant. In this manner, genetically modified, i.e.transgenic or transformed, plants, plant cells, plant tissue, seed,root, and the like can be obtained.

Microbial or Bacterial Cells

The production of bacteria containing heterologous phage resistancegenes, the preparation of starter cultures of such bacteria, and methodsof fermenting substrates, particularly food substrates such as milk, maybe carried out in accordance with known techniques.

By “introducing” as it pertains to nucleic acids is meant introductioninto prokaryotic or eukaryotic cells via conventional transformation ortransfection techniques, or by phage-mediated infection. As used herein,the terms “transformation,” “transduction,” “conjugation,” and“protoplast fusion” are meant to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a cell,including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, or electroporation.Suitable methods for transforming or transfecting cells can be found inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and otherlaboratory manuals. By “introducing” as it pertains to polypeptides ormicroorganisms of the invention, is meant introduction into a host byingestion, topical application, nasal, suppository, urogenital, or oralapplication of the polypeptide or microorganism.

Bacterial cells used to produce the carbohydrate utilization-related ormultidrug transporter polypeptides of this invention are cultured insuitable media, as described generally in Sambrook et al. (1989)Molecular Cloning, A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.

Transgenic Plants and Plant Cells

As used herein, the terms “transformed plant” and “transgenic plant”refer to a plant that comprises within its genome a heterologouspolynucleotide. Generally, the heterologous polynucleotide is stablyintegrated within the genome of a transgenic or transformed plant suchthat the polynucleotide is passed on to successive generations.

The heterologous polynucleotide may be integrated into the genome aloneor as part of a recombinant expression cassette. It is to be understoodthat as used herein the term “transgenic” includes any cell, cell line,callus, tissue, plant part, or plant the genotype of which has beenaltered by the presence of heterologous nucleic acid including thosetransgenics initially so altered as well as those created by sexualcrosses or asexual propagation from the initial transgenic. The term“transgenic” as used herein does not encompass the alteration of thegenome (chromosomal or extra-chromosomal) by conventional plant breedingmethods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct, including a nucleic acid expression cassettethat comprises a transgene of interest, the regeneration of a populationof plants resulting from the insertion of the transgene into the genomeof the plant, and selection of a particular plant characterized byinsertion into a particular genome location. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic makeup of a plant. The term “event” alsorefers to progeny produced by a sexual outcross between the transformantand another variety that includes the heterologous DNA.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants within the scope of theinvention are to be understood to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, stems, fruits,ovules, leaves, or roots originating in transgenic plants or theirprogeny previously transformed with a DNA molecule of the invention, andtherefore consisting at least in part of transgenic cells.

As used herein, the term “plant cell” includes, without limitation,seeds suspension cultures, embryos, meristematic regions, callus tissue,leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza saliva), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Oleo europaea), papaya (Carica papaya), cashew (Anacardiumoccidentals), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca saliva), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

The methods of the invention do not depend on a particular method forintroducing a nucleotide construct to a plant, only that the nucleotideconstruct gains access to the interior of at least one cell of theplant. Methods for introducing nucleotide constructs into plants areknown in the art including, but not limited to, stable transformationmethods, transient transformation methods, and virus-mediated methods.

By “transient transformation” it is meant that a nucleotide constructintroduced into a plant does not integrate into the genome of the plant.By “stable transformation” it is meant that the nucleotide constructintroduced into a plant integrates into the genome of the plant and iscapable of being inherited by progeny thereof. “Primary transformant”and “T0 generation” transgenic plants are of the same genetic generationas the tissue that was initially transformed (i.e., not having gonethrough meiosis and fertilization since transformation). “Secondarytransformants” and “T1, T2, T3, and subsequent generations” refer totransgenic plants derived from primary transformants through one or moremeiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Thenucleotide constructs of the invention may be introduced into plants byany method known in the art, including, but not limited to, contactingthe plants with a virus or viral nucleic acids (see, for example, U.S.Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931;herein incorporated by reference), microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Nail. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,981,840 and 5,563,055), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; and 5,932,782); all of which are hereinincorporated by reference.

The transformed cells may be grown into plants with methods known in theart. See, for example, McCormick et al. (1986) Plant Cell Reports5:81-84. These plants may then be grown, and either pollinated with thesame transformed strain or different strains, and the resulting hybridhaving expression of the desired phenotypic characteristic identified.Two or more generations may be grown to ensure that expression of thedesired phenotypic characteristic is stably maintained and inherited andthen seeds may be harvested to ensure expression of the desiredphenotypic characteristic has been achieved. Thus as used herein,“transformed seeds” refers to seeds that contain the nucleotideconstruct stably integrated into the plant genome.

Methods of Use

Methods are provided for modifying expression of carbohydrateutilization-related or multidrug transporter genes or proteins of anorganism. In one embodiment, properties of microorganisms used infermentation are modified to provide strains able to utilize alternativecarbohydrates for energy or carbon sources. These modifications mayresult in a new ability to synthesize, transport, accumulate, or degradea carbohydrate. Alternatively, these modifications may result in theability to survive contact with antimicrobial polypeptides, includingantibiotics and toxins. These new abilities may also allow themicroorganisms to better survive stressful conditions, such as thedigestive tract or those found during food processing and storage, whichwill increase the utility of these microorganisms in fermenting variousfoods, as well as allowing them to provide longer-lasting probioticactivity after ingestion. These new abilities may also allow themicroorganisms to generate different flavors or textures in a productupon fermentation. In addition, the new abilities may enable a bacteriumto produce a modified carbohydrate, exopolysaccharide, or cell surfacepolysaccharide. In another embodiment, the properties of plants aremodified to provide similar abilities. These abilities are provided bythe nucleotide and amino acid sequences disclosed in the presentinvention.

In general, the methods comprise introducing or overexpressing one ormore proteins involved in carbohydrate utilization or multidrugresistance. By “introducing” is meant that the protein of interest isexpressed in a modified cell when it was not expressed in an unmodifiedcell. By “overexpressing” is meant that the protein of interest isexpressed in an increased amount in the modified organism compared toits production in the unmodified wild-type organism. Homofermentativelactic acid bacteria, in particular, have a relatively simplemetabolism, with almost no overlap between energy metabolism andbiosynthesis metabolism, making them ideal targets for metabolicengineering (Hugenholz and Kleerebezem (1999) Current Opin. Biotech.10:492-497). The expression of bacterial genes in plants is well knownin the art. See, for example, Shewmaker et al. (1994) Plant Physiol.104:1159-1166; Shen et al. (1997) Plant Physiol. 113:1177-1183;Blaszczyk et al. (1999) Plant J. 20:237-243.

Expression of one or more carbohydrate utilization-related or multidrugtransporter proteins may allow for an organism to have a modifiedability to transport a carbohydrate or an antimicrobial polypeptide suchas a bacteriocin into or out of a cell.

Transport-related carbohydrate utilization proteins or multidrugtransporter proteins comprise ABC transporter system componentsincluding substrate-binding proteins (for example HisJ and MalE),membrane-associated components such as permeases (for example LacF andLacG), and cytoplasmic proteins such as ATP-binding proteins (forexample msmK). Transport-related carbohydrate utilization proteins ormultidrug transporter proteins also comprise secondary transport systemproteins such as those in the major facilitator superfamily (MFS) andthe glycoside/pentoside/hexuronide family. Group translocation systemproteins are also included, including enzyme I, enzyme II, and HPrproteins.

Methods are known in the art for cloning and expressing carbohydrateutilization-related proteins in microorganisms and plants, and forassessing function of those proteins (see, for example, de Vos (1996)Antonio van Leeuwenhoek 70:223-242; Yeo et al. (2000) Mol. Cells.10:263-268; Goddijn et al. (1997) Plant Physiol. 113:181-190). Functionfor primary and secondary transport system-related proteins may beassessed, for example, by enzyme assays, fermentation assays, andtransport assays. Function for group translocation system-relatedproteins may be assessed, for example, by sugar phosphorylation assays.See, for example, Russell et al. (Russell et al. (1992) J. Biol. Chem.267:4631-4637), where genes from a primary transport system (msm) inStreptococcus mutans are identified and expressed in E. coli;Leong-Morgenthaler et al. (Leong-Morgenthaler et al. (1991) J.Bacteriol. 173:1951-1957, where two genes from a secondary transportsystem (lactose) from Lactobacillus bulgaricus were cloned and expressedin E. coli; Vaughan et al. (Vaughan et al. (1996) Appl. Env. Microbiol.62:1574-1582), where a secondary transport system (lacS) gene fromLeuconostoc lactis was cloned and expressed in E. coli; de Vos et al.(de Vos et al. (1990) J. Biol. Chem. 265:22554-22560), where two PTSsystem genes from Lactococcus lactis were identified, cloned andexpressed in E coli and Lactobacillus lactis; Sato et al. (Sato et al.(1989) J. Bacteriol. 171:263-271), where the scrA gene fromStreptococcus mutans was cloned into E. coli and found to exhibitsucrose PTS activity; Alpert and Chassy (Alpert and Chassy (1990) J.Biol. Chem. 265:22561-22568), where the gene coding for thelactose-specific Enzyme II of Lactobacillus casei was cloned andexpressed in E. coli; Boyd et al. (Boyd et al. (1994) Infect. Immun.62:1156-1165), where the genes that encode HPr and Enzyme I of the PTStransport system of Streptococcus mutans were cloned and expressed in E.coli; Garg et al. (Garg et al. (2002) Proc. Natl. Acad. Sci. USA99:15898-15903), where the overexpression of E. coli trehalosebiosynthetic genes otsA and otsB led to increased tolerance of thetransgenic plants to abiotic stress, and enhanced productivity; andGrinius and Goldberg (Grinius and Goldberg (1994)J. Biol. Chem.269:29998-30004), where a multidrug resistance protein was expressed anddemonstrated to function as a drug pump.

Expression of one or more carbohydrate utilization-related proteins mayallow for an organism to have a modified ability to accumulate acarbohydrate in the cytoplasm of a cell. For example, introducing oroverexpressing an enzyme involved in sugar catabolism without expressinga relevant transport protein may lead to an accumulation of thatcarbohydrate in the cytoplasm. Alternatively, introduction oroverexpression of a carbohydrate transport-related protein may lead toenhanced transport of the carbohydrate into the external environment.Methods are known in the art for introducing or expressingcarbohydrate-related genes in organisms. Accumulation of a carbohydratein a cell may be assessed, for example, by chromatographic methods orenzyme assays. See, for example, Chaillou et al. (1998) J. Bacteriol.180:4011-4014 and Goddijn et al. (1997) supra.

Expression of one or more carbohydrate utilization-related proteins mayallow for an organism to have a modified ability to utilize or produce acarbohydrate as an energy source. Methods are known in the art forcloning and expressing carbohydrate utilization-related proteins inorganisms, and for assessing function of those proteins (see, forexample, de Vos (1996) Anionic van Leeuwenhoek 70:223-242; Hugenholz etal. (2002) Antonie van Leeuwenhoek 82:217-235). For example, the genesfor lactose metabolism may be introduced into a bacterium to improve theutilization of lactose, and to produce a product more acceptable tolactose-intolerant people (Hugenholz et al. (2002) supra). Furthermodifications may be made in these modified bacteria, such as blockingglucose metabolism so that glucose is not degraded, but is released fromthe cell into the medium, thereby providing natural sweetness. See, forexample (Hugenholz et al. (2002) supra). Alternatively, the genes forgalactose metabolism as well as the gene for α-phosphoglucomutase may beintroduced, to improve the galactose-fermenting capability of themicroorganism, thereby aiding in preventing the consumption of highlevels of galactose, which could cause health problems (Hugenholz et al.(2002) supra; Hirasuka and Li (1992) J. Stud. Alcohol 62:397-402). Onegene associated with galactose metabolism is α-galactosidase, theexpression of which may be useful for removing raffinose-type sugarsfrom fermented products, since monogastric animals cannot degrade them(Hugenholz et al. (2002) supra). Expression of the bacterial gene formannitol-1-phosphate dehydrogenase (mtlD) in tobacco plants successfullyresulted in the synthesis and accumulation of mannitol (Tarczynski etal. (1992) Proc. Natl. Acad. Sci. USA 89:2600-2604).

Function of the various carbohydrate-related proteins may be assessed,for example, by microbial growth assays, transport assays, enzymeassays, or analysis by chromatography methods and NMR. See, for example,Djordjevic et al. (2001) J. Bacteriol. 183:3224-3236; Chaillou et al.(1998) J. Bacteriol. 180:4011-4014; and Tarczynski et al. (1992) supra.

Generally, permeases, membrane-associated enzymes, and regulators suchas transcriptional repressors or antiterminators may need to beexpressed in the cell for optimal utilization of a carbohydrate. Thefunction of transcriptional antiterminators may be assayed byantitermination activity in a reporter system (see, for example, Alpertand Siebers (1997) J. Bacteriol. 179:1555-1562). The function ofrepressors such as lacR may be assessed by enzyme activity or growthassays (see, for example, van Rooijen et al. (1993) Protein Eng.6:201-206; van Rooijen and de Vos (1990) J. Biol. Chem.265:18499-18503).

The sequences of the present invention may also modify the ability of anorganism to alter the flavor or texture of a food product. Modificationof glucose metabolism to produce alternative sugars is one approach thatmay lead to altered flavor or textural characteristics. Disruption ofthe lactate dehydrogenase gene with the concomitant expression of genesfrom the mannitol or sorbitol operons results in the production ofmannitol and sorbitol (Hugenholz et al. (2002) supra). Diacetylproduction during fermentation results in a butter aroma, which can beenhanced by either disruption of lactate dehydrogenase or overexpressionof NADH oxidase in combination with disruption of α-acetolactatedecarboxylase (Hugenholz and Kleerebezem, (1999) supra; Hugenholtz etal. (2000) Appl. Environ. Microbiol. 66:4112-4114) Alternatively,overproduction of α-acetolactate synthase or acetohydroxy acid synthasewith disruption of α-acetolactate decarboxylase has resulted inincreased diacetyl production (Swindell et al. (1996) Appl. Environ.Microbiol. 62:2641-2643; Platteeuw et al. (1995) Appl. Environ.Microbiol. 61:3967-3971). Overexpression of alanine dehydrogenaseresults in the production of alanine instead of lactic acid, providing ataste-enhancer and sweetener in fermented foods (Hols et al. (1999) Nat.Biotechnol. 17:588-592).

Methods for modifying the ability of an organism to produce a modifiedcarbohydrate are also encompassed, comprising introducing at least onenucleotide sequence of the present invention into an organism. Methodsfor producing modified carbohydrates are also encompassed, and comprisecontacting a carbohydrate to be modified with a polypeptide of thepresent invention. Methods are known in the art for producing modifiedcarbohydrates. See, for example Kim et al. (2001) Biotechnol. Frog.17:208-210.

The sequences of the current invention may also modify the ability of anorganism to survive in a food system or the gastrointestinal tract of amammal, or modify an organism's stability and survival during foodprocessing and storage. For example, increased production of trehalosemay result in prolonged freshness and taste of a fermented product (see,for example, www.nutracells.com). Trehalose also may aid in theprevention of diseases that result from protein aggregation orpathological conformations of proteins, such as Creutzfeld-Jacobdisease. In plants, accumulation of trehalose leads to protectionagainst environmental stresses such as drought, salt, and cold (see, forexample, Jang et al. (2003) Plant Physiol. 131:516-524; Penna (2003)Trends Plant Sci. 8:355-357; Garg et al. (2002) Proc. Natl. Acad.Science 99:15898-15903; Yeo et al. (2000) supra). In addition, plantshave been transformed with fructosyltransferase genes, which enabled theplant to accumulate fructans to a high level (van der Meer et al. (1994)Plant Cell 6:561-570). In addition to having a role as a carbohydratereserve, fructans may also provide tolerance to dry and cold conditions(Pontis and del Campillo (1985) “Fructans” in Biochemistry of StorageCarbohydrates in Green Plants, Day and Dixon, eds. (London: AcademicPress), pp. 810-816; Pilon-Smits et al. (1995) Plant Physiol.107:125-130). The bacterial gene mannitol-1-phosphate dehydrogenase hasalso been expressed in plants, resulting in the production of mannitol,which is thought to confer beneficial traits including osmoregulationand neutralization of hydroxyl radicals (Tarczynski et al. (1992)supra).

The multidrug transporter sequences of the invention may allow anorganism to survive contact with an antimicrobial polypeptide or othertoxin. This may be due to an increased ability to transport a drug ortoxin out of the cell.

Variants of these nucleotide sequences are also encompassed, such asthose that retain or modify the ability to transport a carbohydrate ortoxin into or out of a cell, and those that retain or modify the abilityto accumulate or utilize a carbohydrate. Methods for making and testingvariants of carbohydrate utilization-related or multidrug transporterproteins are well known in the art. See, for example, Poolman et al.(Poolman et al. (1996) Mol. Microbiol. 19:911-911), where variants ofsecondary transport system proteins (mellibiose and lactose) withaltered substrate specificities were isolated or constructed and tested.In these mutants, sugar transport is uncoupled from cation symport. Seealso, for example, Djorovevic et al. (2001) supra, where mutant HPrproteins were constructed with altered regulatory activity; and Adams etal. (Adams et al. (1994) J. Biol. Chem. 269:5666-5672), wherecold-sensitive variants of the β-galactosidase gene from Lactobacillusdelbrüeckii subsp. bulgaricus were generated and characterized. Thesemutated genes had a reduced V_(max) at low temperatures and thereforemay be useful in preventing the acidification of fermented productsduring cold storage (Mainzer et al. (1990) “Pathway engineering ofLactobacillus bulgaricus for improved yoghurt,” in Yoghurt: Nutritionaland Health Properties, Chandan, ed., (National Yoghurt Association,Virginia, US), pp. 41-55. See, also, Bettenbrock et al. (Bettenbrock etal. (1999) J. Bacteriol. 181:225-230), where mutants with modifiedgalactose-specified PTS genes were isolated. See also, van Rooijen etal. (1990) supra, where variants of the lacR repressor were isolatedthat had no effect on activity. See also Kroetz et al., wherepolymorphism of the human MDR1 gene was analyzed (Kroetz et al. (2003)Pharmacogenetics 13:481-94), and Mitomo et al., where variants of theABC transporter ABCG2 were analyzed (Mitomo et al. (2003) Biochem. J.373:767-74).

Any of the above modifications may be combined with other metabolicalterations that have been engineered or suggested in lactic acidbacteria. These include, B-vitamin production, such as folate (B11),riboflavin (B2), or cobalamin (B12), the production of polyols, orlow-calorie sugars, that could replace sucrose, lactose, glucose, orfructose as sweeteners, the production of tagatose, another sucrosereplacement, the production of various exopolysaccharides, blockingglucose metabolism to provide a natural sweetening effect, reducedproduction of galactose, production of foods with lower levels ofα-galactosides such as stachyose and raffinose, and increased productionof trehalose, which has preserving properties for foodstuffs and ispotentially involved in disease prevention (Hugenholz et al. (2002)supra; van Roojen et al. (1991) J. Biol. Chem. 266:7176-7178).

Methods are also provided for eliminating or modifying undesirablecarbohydrates from a food or chemical product. The methods comprisecontacting the product with a purified polypeptide of the presentinvention. Methods to assay for the elimination or modification ofcarbohydrates are well known in the art.

TABLE 1 Carbohydrate Utilization Proteins of the Present Invention SEQORF # ID NO: IDENTITY/FUNCTION 452 1, 2 PTS system mannose-specificfactor IIAB 877 3, 4 Phosphotransferase system (PTS) lichenan-specificenzyme IIA component 609 5, 6 Beta-glucoside specific transport protein1479 7, 8 Transcription antiterminator 1574  9, 10Phospho-beta-glucosidase 1707 11, 12 Beta-glucoside permease IIABCcomponent 725 13, 14 PTS system, beta-glucosides-specific IIABCcomponent 491 15, 16 Phosphotransferase system (PTS) protein, lichenan-specific enzyme IIC component 1369 17, 18 Phosphotransferase systemenzyme II 1684 19, 20 Phosphotransferase system IIA component 146 21, 22PTS system enzyme IIBC component (galactitol/fructose-specific) 227 23,24 PTS cellobiose-specific component IIC 989 25, 26 PTScellobiose-specific enzyme IIC 884 27, 28 Cellobiose-specific PTS systemIIC component 618 29, 30 PTS system, cellobiose-specific enzyme IIC 60631, 32 Phosphotransferase system (PTS) arbutin-like enzyme IIBCcomponent 1705 33, 34 Sucrose-specific PTS system IIBC component 177735, 36 PTS system protein 500 37, 38 Sucrose operon repressor 502 39, 40ABC transporter substrate-binding protein 503 41, 42 ABC transportermembrane-spanning permease— sugar transporter 504 43, 44 ABC transportermembrane-spanning permease— sugar transport protein 505 45, 46Sucrose-6-phosphate hydrolase 506 47, 48 Multiple sugar-bindingtransport ATP-binding protein 507 49, 50 gtfA protein 1481 51, 52 RiboseABC transporter(ribose-binding protein) 1482 53, 54 Ribose ABCtransporter (permease) 1483 55, 56 Ribose ABC transporter ATP bindingprotein 1484 57, 58 Ribose permease (RbsD) 1485 59, 60 Ribokinase (RbsK)1864 61, 62 Maltose ABC transporter permease protein 1865 63, 64 MaltoseABC transporter permease protein 1866 65, 66 Maltose ABC transportersubstrate binding protein 1867 67, 68 Multiple sugar-binding transportATP-binding protein 1944 69, 70 Sugar ABC transporter protein 1945 71,72 Sugar ABC transporter permease protein 1946 73, 74 Sugar ABCtransporter permease protein 45 75, 76 Sugar transporter 552 77, 78Transporter protein 566 79, 80 Transporter protein 567 81, 82Drug-efflux transporter 753 83, 84 Transporter protein 1446 85, 86Drug-export protein 1471 87, 88 Efflux protein 1616 89, 90 Transporterprotein 1621 91, 92 Efflux transporter protein 1853 93, 94 Drug-effluxtransporter protein 1917 95, 96 Polysaccharide transporter 399 97, 98Sucrose operon regulatory protein 400  99, 100 Sucrose-6-phosphatehydrolase 401 101, 102 Phosphotransferase system enzyme II 1012 103, 104Beta-glucoside-specific PTS system IIABC component 1013 105, 106Trehalose operon transcriptional repressor 1014 107, 108 Dextranglucosidase 1439 109, 110 ABC transporter ATP-binding protein—multiplesugar Transport 1440 111, 112 Multiple sugar-binding transport systempermease protein 1441 113, 114 ABC transporter membrane-spanningpermease— Multiple sugars 1442 115, 116 Multiple sugar-binding proteinprecursor 1443 117, 118 Raffinose operon transcriptional regulatoryprotein 73 119, 120 Carbohydrate-utilization-related 74 121, 122 ABCtransporter bacteriocin 75 123, 124 ABC transporter 1131 125, 126 ABCtransporter 1132 127, 128 ABC transporter 1357 129, 130 ABC transporter1358 131, 132 ABC transporter 1679 133, 134 Permease 1680 135, 136Transporter 1681 137, 138 Carbohydrate-utilization-related 1793 139, 140Carbohydrate-utilization-related 1794 141, 142Carbohydrate-utilization-related 1796 143, 144 plnG 1838 145, 146 ABCtransporter 1839 147, 148 Permease 1840 149, 150 Regulator 1913 151, 152ABC transporter 1914 153, 154 ABC transporter 1915 155, 156Carbohydrate-utilization-related 1938 157, 158Carbohydrate-utilization-related 1939 159, 160 ABC transporter 453 161,162 Mannose-specific phosphotransferase system component 454 163, 164PTS system mannose-specific factor IIAB 455 165, 166 PTS systemmannose-specific, factor IIC 456 167, 168 PTS system mannose-specificfactor IID 876 169, 170 PTS system enzyme II protein 879 171, 172Phosphotransferase system sugar-specific EII component 1575 173, 174 PTSsystem, beta-glucoside-specific enzyme II, ABC component 1463 175, 176LacS 639 177, 177 ptsH 640 179, 180 ptsI 431 181, 182 ccpA 676 183, 184ptsK 1778 185, 186 FruK 1779 187, 188 FruR 1433 189, 190dihydroxyacetone kinase 1434 191, 192 dihydroxyacetone kinase 1436 193,194 glycerol uptake facilitator 1437 195, 196 gtfAII 1438 197, 198 melA1457 199, 200 GalM 1458 201, 202 GalT 1459 203, 204 GalK 1460 205, 206surface protein 1461 207, 208 conserved hypothetical protein 1462 209,210 LacZ 1467 211, 212 beta-galactosidase 1468 213, 214beta-galactosidase 1469 215, 216 GalE 1719 217, 218 UDP-glucosephosphorylase 874 219, 220 beta-galactosidase 910 221, 222 L-LDH 1007223, 224 pyridoxal kinase 1812 225, 226 alpha-glucosidase 1632 227, 228aldehyde dehydrogenase 1401 229, 230 NADH peroxidase 1974 231, 232pyruvate oxidase 1102 233, 234 amino acid permease 1783 235, 236 ABCtransporter 1879 237, 238 pyrimidine kinase 680 239, 240 glgB 55 241,242 D-LDH 185 243, 244 phosphoglycerate mutase 271 245, 246 L-LDH 698247, 248 GPDH 699 249, 250 phosphoglycerate kinase 752 251, 252 glucose6-phosphate isomerase 889 253, 254 2-phosphoglycerate dehydratase 956255, 256 phosphofructokinase 957 257, 258 pyruvate kinase 1599 259, 260fructose bisphosphate aldolase 1641 261, 262 glycerol 3-phosphate ABCtransporter 452 263, 264 Mannose; PTS system mannose-specific factorIIAB 1479 265, 266 beta-glucoside; transcription antiterminator 725 267,268 beta-glucoside; PTS system, beta-glucosides- specific IIABCcomponent 1369 269, 270 Cellobiose; phosphotransferase system enzyme II227 271, 272 Cellobiose; PTS cellobiose-specific component II 502 273,274 sugar transporter; ABC transporter substrate-binding protein 507275, 276 GtfA 1483 277, 278 rbsA; ribose ABC transporter ATP bindingprotein 1484 279, 280 ribose permease RbsD 552 281, 282 multidrugtransporter 567 283, 284 multidrug transporter 1471 285, 286 multidrugtransporter 1853 287, 288 multidrug transporter 1012 289, 290 treB;beta-glucoside; beta-glucoside-specific PTS system IIABC component 1014291, 292 treC 1440 293, 294 msmG 1442 295, 296 msmE 1132 297, 298 ABCtransporter 1358 299, 300 ABC transporter 1838 301, 302 ABC transporter1840 303, 304 transcriptional regulator (TetR/AcrR family) 1913 305, 306ABC transporter 1938 307, 308 164 309, 310 multidrug transporter 251311, 312 multidrug transporter 252 313, 314 multidrug transporter 253315, 316 multidrug transporter 1062 317, 318 multidrug transporter 597319, 320 ABC multidrug transporter 1854 321, 322 multidrug transporter

The following examples are offered by way of illustration and not by wayof limitation.

Example 1 Gapped BlastP Results for Amino Acid Sequences

A Gapped BlastP sequence alignment showed that SEQ ID NO:2 (144 aminoacids) has about 61% identity from amino acids 1-140 with a protein fromListeria innocua that is homologous to a PTS system mannose-specificfactor IIAB (Accession Nos. NP_(—)469488.1; NC_(—)003212), about 60%identity from amino acids 1-140 with a protein from Listeriamonocytogenes that is homologous to a PTS system mannose-specific factorIIAB (Accession Nos. NP_(—)463629.1; NC_(—)003210), about 63% identityfrom amino acids 1-139 with a protein from Clostridium acetobutylicumthat is a mannose-specific phosphotransferase system component IIAB(Accession Nos. NP_(—)149230.1; NC_(—)001988), about 62% identity fromamino acids 1-139 with a protein from Clostridium perfringens that is aPTS system protein (Accession Nos. NP_(—)561737.1; NC_(—)003366), andabout 50% identity from amino acids 2-141 with a protein fromStreptococcus pyogenes that is a mannose-specific phosphotransferasesystem component IIAB (Accession Nos. NP_(—)269761.1; NC_(—)002737).

A Gapped BlastP sequence alignment showed that SEQ ID NO:4 (123 aminoacids) has about 60% identity from amino acids 20-109 with a proteinfrom Listeria innocua that is homologous to a phosphotransferase system(PTS) lichenan-specific enzyme IIA component (Accession Nos.NP_(—)471165.1; NC_(—)003212), about 57% identity from amino acids20-110 with a protein from Listeria innocua that is homologous to acellobiose phosphotransferase enzyme IIA component (Accession Nos.NP_(—)472161.1; NC_(—)003212), about 46% identity from amino acids 1-112with a protein from Lactococcus lactis subsp. lactis that is acellobiose-specific PTS system IIA component (EC 2.7.1.69) (AccessionNos. NP_(—)266570.1; NC_(—)002662), about 44% identity from amino acids9-112 with a protein from Bacillus halodurans that is a PTS system,cellobiose-specific enzyme IIA component (Accession Nos. NP_(—)241776.1;NC_(—)002570), and about 51% identity from amino acids 16-112 with aprotein from Streptococcus pyogenes that is homologous to a PTS enzymeIII (Accession Nos. NP_(—)607437.1; NC_(—)003485).

A Gapped BlastP sequence alignment showed that SEQ ID NO:6 (161 aminoacids) has about 53% identity from amino acids 6-143 with a protein fromEnterococcus faecium that is a beta-glucoside specific transport protein(BglS) (Accession Nos. gb|AAD28228.1; AF121254), about 48% identity fromamino acids 13-159 with a protein from Streptococcus pneumoniae that isa PTS system, IIABC component (Accession Nos. NP_(—)345256.1;NC_(—)003028), about 48% identity from amino acids 13-159 with a proteinfrom Streptococcus pneumoniae that is a PTS glucose-specific enzymeIIABC component (Accession Nos. NP_(—)358262.1; NC_(—)003098), about 46%identity from amino acids 13-159 with a protein from Streptococcuspyogenes that is homologous to a PTS system, enzyme IIA component(Accession Nos. NP_(—)608025.1; NC_(—)003485), and about 46% identityfrom amino acids 13-159 with a protein from Streptococcus pyogenes thatis homologous to a PTS system, enzyme IIA component (Accession Nos.NP_(—)269950.1; NC_(—)002737).

A Gapped BlastP sequence alignment showed that SEQ ID NO:8 (291 aminoacids) has about 36% identity from amino acids 11-282 with a proteinfrom Bacillus subtilis that is a transcription antiterminator (licT)(Accession No. sp|P39805|LICT_BACSU), about 36% identity from aminoacids 11-282 with a protein from Bacillus subtilis that is atranscriptional antiterminator (BglG family) (Accession Nos.NP_(—)391787.1; NC_(—)000964), about 37% identity from amino acids11-282 with a protein from Escherichia coli that is involved in positiveregulation of the bgl operon (Accession Nos. NP_(—)418179.1;NC_(—)000913), about 33% identity from amino acids 11-282 with a proteinfrom Erwinia chrysanthemi that is a beta-glucoside operon antiterminator(Accession No. sp|P26211|ARBG_ERWCH), and about 34% identity from aminoacids 9-288 with a protein from Clostridium acetobutylicum that is atranscriptional antiterminator (licT) (Accession Nos. NP_(—)347062.1;NC_(—)003030).

A Gapped BlastP sequence alignment showed that SEQ ID NO:10 (480 aminoacids) has about 59% identity from amino acids 8-473 with a protein fromListeria monocytogenes that is homologous to a phospho-beta-glucosidase(Accession Nos. NP_(—)463849.1; NC_(—)003210), about 58% identity fromamino acids 8-473 with a protein from Listeria innocua that ishomologous to a phospho-beta-glucosidase (Accession Nos. NP_(—)469689.1;NC_(—)003212), about 57% identity from amino acids 7-473 with a proteinfrom Clostridium acetobutylicum that is a 6-phospho-beta-glucosidase(NP_(—)347379.1; NC_(—)003030), about 57% identity from amino acids8-473 with a protein from Clostridium longisporum that is a6-phospho-beta-glucosidase (Accession No. sp|Q46130|ABGA_CLOLO), andabout 55% identity from amino acids 1-473 with a protein from Bacillussubtilis that is a beta-glucosidase (Accession Nos. NP_(—)391805.1;NC_(—)000964).

A Gapped BlastP sequence alignment showed that SEQ ID NO:12 (625 aminoacids) has about 38% identity from amino acids 1-624 with a protein fromStreptococcus pyogenes that is a beta-glucoside permease IIABC component(Accession Nos. NP_(—)268836.1; NC_(—)002737), about 38% identity fromamino acids 1-624 with a protein from Streptococcus pyogenes that is abeta-glucoside permease IIABC component (Accession Nos. NP_(—)606826.1;NC_(—)003485), about 38% identity from amino acids 1-605 with a proteinfrom Streptococcus pneumoniae that is a phosphotransferase systemsugar-specific EII component (Accession Nos. NP_(—)358099.1;NC_(—)003098), about 38% identity from amino acids 1-605 with a proteinfrom Streptococcus pneumoniae that is a PTS system,beta-glucosides-specific IIABC component (Accession Nos. NP_(—)345091.1;NC_(—)003028), and about 38% identity from amino acids 1-622 with aprotein from Bacillus halodurans that is a PTS system,beta-glucoside-specific enzyme IIABC component (Accession Nos.NP_(—)241162.1; NC_(—)002570).

A Gapped BlastP sequence alignment showed that SEQ ID NO:14 (675 aminoacids) has about 50% identity from amino acids 17-648 with a proteinfrom Clostridium acetobutylicum that is a PTS system,beta-glucosides-specific IIABC component (Accession Nos. NP_(—)348035.1;NC_(—)003030), about 50% identity from amino acids 17-656 with a proteinfrom Bacillus halodurans that is a PTS system, beta-glucoside-specificenzyme IIABC (Accession Nos. NP_(—)241461.1; NC_(—)002570), about 50%identity from amino acids 17-656 with a protein from Listeriamonocytogenes that is homologous to a PTS system, beta-glucosidesspecific enzyme IIABC (Accession Nos. NP_(—)463560.1; NC_(—)003210),about 48% identity from amino acids 17-654 with a protein fromClostridium longisporum that is a PTS-dependent enzyme II (AccessionNos. gb|AAC05713.1; L49336), and 48% identity from amino acids 13-654with a protein from Streptococcus mutans that is abeta-glucoside-specific EII permease (Accession Nos. gb|AAF89975.1;AF206272). A Gapped BlastP sequence alignment showed that SEQ ID NO:16(445 amino acids) has about 41% identity from amino acids 10-443 with aprotein from Bacillus subtilis that is a phosphotransferase system (PTS)protein, lichenan-specific enzyme IIC component (Accession Nos.NP_(—)391737.1; NC_(—)000964), about 42% identity from amino acids14-442 with a protein from Bacillus subtilis that is homologous to a PTSsystem IIBC component (ywbA) (Accession No. sp|P39584|YWBA_BACSU), about41% identity from amino acids 14-441 with a protein from Bacillusstearothermophilus that is a cellobiose phosphotransferase enzyme IICcomponent (Accession No. sp|Q45400|PTCC_BACST), about 41% identity fromamino acids 12-441 with a protein from Streptococcus pneumoniae that isa phosphotransferase system sugar-specific EII component (Accession Nos.NP_(—)358015.1; NC_(—)003098), and 40% identity from amino acids 12-441with a protein from Streptococcus pneumoniae that is a PTS system,cellobiose-specific IIC component (Accession Nos. NP_(—)344993.1;NC_(—)003028).

A Gapped BlastP sequence alignment showed that SEQ ID NO:18 (422 aminoacids) has about 34% identity from amino acids 9-417 with a protein fromBacillus subtilis that is homologous to a phosphotransferase systemenzyme II (Accession Nos. NP_(—)391718.1; NC_(—)000964), about 33%identity from amino acids 17-414 with a protein from Bacillus subtilisthat is a phosphotransferase system (PTS) lichenan-specific enzyme IICcomponent (Accession Nos. NP_(—)391737.1; NC_(—)000964), about 34%identity from amino acids 10-417 with a protein from Bacillusstearothermophilus that is a cellobiose phosphotransferase enzyme IICcomponent (Accession No. sp|Q45400|PTCC_BACST), about 33% identity fromamino acids 9-414 with a protein from Listeria innocua that ishomologous to a PTS system, cellobiose-specific IIC component (AccessionNos. NP_(—)470241.1; NC_(—)003212), and 31% identity from amino acids11-415 with a protein from Borrelia burgdorferi that is a PTS system,cellobiose-specific TIC component (celB) (Accession Nos. NP_(—)046990.1;NC_(—)001903).

A Gapped BlastP sequence alignment showed that SEQ ID NO:20 (130 aminoacids) has about 33% identity from amino acids 3-124 with a protein fromBrucella melitensis that is a phosphotransferase system IIA component(Accession Nos. NP_(—)540949.1; NC_(—)003317), about 32% identity fromamino acids 2-102 with a protein from Lactobacillus curvatus that is anEIIA-mannose protein (Accession Nos. gb|AAB04153.1; U28163), about 32%identity from amino acids 3-96 with a protein from Clostridiumperfringens that is homologous to a PTS system protein (Accession Nos.NP_(—)563545.1; NC_(—)003366), about 25% identity from amino acids 3-123with a protein from Clostridium perfringens that is homologous to a PTSsystem protein (Accession Nos. NP_(—)561737.1; NC_(—)003366), and 25%identity from amino acids 3-123 with a protein from Clostridiumacetobutylicum that is a mannose-specific phosphotransferase systemcomponent IIAB (Accession Nos. NP_(—)149230.1; NC_(—)001988).

A Gapped BlastP sequence alignment showed that SEQ ID NO:22 (162 aminoacids) has about 38% identity from amino acids 8-159 with a protein fromClostridium acetobutylicum that is a PTS system enzyme IIBC component(galactitol/fructose-specific) (Accession Nos. NP_(—)349560.1;NC_(—)003030), about 36% identity from amino acids 7-158 with a proteinfrom Streptococcus pneumoniae that is a phosphotransferase systemsugar-specific Eli component (Accession Nos. NP_(—)358156.1;NC_(—)003098), about 36% identity from amino acids 7-158 with a proteinfrom Streptococcus pneumoniae that is homologous to a PTS system IIAcomponent (Accession Nos. NP_(—)345152.1; NC_(—)003028), about 38%identity from amino acids 20-134 with a protein from Streptococcusagalactiae that is a GatA protein (Accession Nos. gb|AAG09977.1;AF248038), and 33% identity from amino acids 16-159 with a protein fromBacillus halodurans that is a PTS system, galactitol-specific enzyme IIAcomponent (Accession Nos. NP_(—)241058.1; NC_(—)002570).

A Gapped BlastP sequence alignment showed that SEQ ID NO:24 (466 aminoacids) has about 47% identity from amino acids 30-461 with a proteinfrom Clostridium acetobutylicum that is a PTS cellobiose-specificcomponent IIC (Accession NP_(—)347026.1; NC_(—)003030), about 45%identity from amino acids 26-465 with a protein from Lactococcus lactissubsp. lactis that is a cellobiose-specific PTS system IIC component (EC2.7.1.69) (Accession Nos. NP_(—)266974.1; NC_(—)002662), about 46%identity from amino acids 82-465 with a protein from Lactococcus lactissubsp. lactis that is a cellobiose-specific PTS system IIC component (EC2.7.1.69) (Accession Nos. NP_(—)266572.1; NC_(—)002662), about 41%identity from amino acids 34-466 with a protein from Streptococcuspyogenes that is homologous to a PTS system, enzyme IIC component(Accession Nos. NP_(—)269994.1; NC_(—)002737), and 40% identity fromamino acids 34-466 with a protein from Streptococcus pyogenes that ishomologous to a PTS system, enzyme IIC component (Accession Nos.NP_(—)608069.1; NC_(—)003485).

A Gapped BlastP sequence alignment showed that SEQ ID NO:26 (428 aminoacids) has about 28% identity from amino acids 25-420 with a proteinfrom Listeria innocua that is homologous to a PTS cellobiose-specificenzyme IIC (Accession NP_(—)472233.1; NC_(—)003212), about 27% identityfrom amino acids 115-415 with a protein from Lactobacillus casei that isa LacE protein (Accession Nos. emb|CAB02556.1; Z80834), about 26%identity from amino acids 137-425 with a protein from Listeria innocuathat is homologous to a PTS system, cellobiose-specific enzyme IIC(Accession Nos. NP_(—)472184.1; NC_(—)003212), about 26% identity fromamino acids 137-425 with a protein from Listeria monocytogenes that ishomologous to a PTS system, cellobiose-specific enzyme IIC (AccessionNos. NP_(—)466230.1; NC_(—)003210), and 26% identity from amino acids115-415 with a protein from Lactobacillus casei that is aphosphotransferase system enzyme II (EC 2.7.1.69) (Accession No.pir∥B23697).

A Gapped BlastP sequence alignment showed that SEQ ID NO:28 (475 aminoacids) has about 57% identity from amino acids 10-471 with a proteinfrom Lactococcus lactis subsp. lactis that is a cellobiose-specific PTSsystem IIC component (EC 2.7.1.69) (Accession Nos. NP_(—)266974.1;NC_(—)002662), about 45% identity from amino acids 71-475 with a proteinfrom Lactococcus lactis subsp. lactis that is a cellobiose-specific PTSsystem IIC component (EC 2.7.1.69) (Accession Nos. NP_(—)266572.1;NC_(—)002662), about 42% identity from amino acids 13-470 with a proteinfrom Clostridium acetobutylicum that is a PTS cellobiose-specificcomponent IIC (Accession Nos. NP_(—)347026.1; NC_(—)003030), about 41%identity from amino acids 17-468 with a protein from Streptococcuspyogenes that is homologous to a PTS system, enzyme IIC component(Accession Nos. NP_(—)269994.1; NC_(—)002737), and 41% identity fromamino acids 17-468 with a protein from Streptococcus pyogenes that ishomologous to a PTS system, enzyme IIC component (Accession Nos.NP_(—)608069.11 (NC_(—)003485).

A Gapped BlastP sequence alignment showed that SEQ ID NO:30 (441 aminoacids) has about 46% identity from amino acids 1-428 with a protein fromListeria innocua that is homologous to a PTS system, cellobiose-specificenzyme IIC (Accession Nos. NP_(—)472184.1; NC_(—)003212), about 46%identity from amino acids 1-428 with a protein from Listeriamonocytogenes that is homologous to a PTS system, cellobiose-specificenzyme IIC (Accession Nos. NP_(—)466230.1; NC_(—)003210), about 39%identity from amino acids 10-427 with a protein from Streptococcuspyogenes that is homologous to a PTS system IIC component (AccessionNos. NP_(—)607435.1; NC_(—)003485), about 36% identity from amino acids1-428 with a protein from Lactococcus lactis subsp. lactis that is acellobiose-specific PTS system IIC component (EC 2.7.1.69) (AccessionNos. NP_(—)266330.1; NC_(—)002662), and 31% identity from amino acids1-421 with a protein from Listeria monocytogenes that is homologous to acellobiose phosphotransferase enzyme IIC component (Accession Nos.NP_(—)466206.1; NC_(—)003210).

A Gapped BlastP sequence alignment showed that SEQ ID NO:32 (626 aminoacids) has about 54% identity from amino acids 1-532 with a protein fromBacillus subtilis that is a phosphotransferase system (PTS) arbutin-likeenzyme IIBC component (Accession Nos. NP_(—)388701.1; NC_(—)000964),about 51% identity from amino acids 2-530 with a protein fromClostridium perfringens that is a PTS arbutin-like enzyme IIBC component(Accession Nos. NP_(—)561112.1; NC_(—)003366), about 52% identity fromamino acids 1-533 with a protein from Fusobacterium mortiferum that is aPTS protein EII (Accession Nos. gb|AAB63014.2; U81185), about 51%identity from amino acids 1-533 with a protein from Clostridiumacetobutylicum that is a MaIP protein (Accession Nos. gb|AAK69555.1;AF290982), and 51% identity from amino acids 1-533 with a protein fromClostridium acetobutylicum that is a PTS system, arbutin-like IIBCcomponent (Accession Nos. NP_(—)347171.1; NC_(—)003030).

A Gapped BlastP sequence alignment showed that SEQ ID NO:34 (663 aminoacids) has about 58% identity from amino acids 1-456 with a protein fromLactococcus lactis subsp. lactis that is a sucrose-specific PTS systemIIBC component (EC2.7.1.69) (Accession Nos. NP_(—)267287.1;NC_(—)002662), about 54% identity from amino acids 5-471 with a proteinfrom Staphylococcus aureus subsp. aureus that is homologous to a sucrosephosphotransferase enzyme II (Accession Nos. NP_(—)373429.1;NC_(—)002745), about 46% identity from amino acids 5-472 with a proteinfrom Bacillus halodurans that is a PTS system, sucrosephosphotransferase enzyme IIBC component (Accession Nos. NP_(—)244441.1;NC_(—)002570), about 39% identity from amino acids 4-468 with a proteinfrom Salmonella enterica subsp. enterica serovar Typhi that ishomologous to a PTS system IIBC component (Accession Nos.NP_(—)457099.1; NC_(—)003198), and 39% identity from amino acids 4-468with a protein from Salmonella typhimurium that is homologous to aphosphotransferase system IIB component (Accession Nos. NP_(—)461505.1;NC_(—)003197).

A Gapped BlastP sequence alignment showed that SEQ ID NO:36 (665 aminoacids) has about 44% identity from amino acids 1-661 with a protein fromClostridium perfringens that is a PTS system protein (Accession Nos.NP_(—)561500.1; NC_(—)003366), about 46% identity from amino acids 1-657with a protein from Streptococcus pyogenes that is homologous to afructose-specific enzyme II, PTS system BC component (Accession Nos.NP_(—)269062.1; NC_(—)002737), about 46% identity from amino acids 1-657with a protein from Streptococcus pyogenes that is homologous to afructose-specific enzyme II, PTS system BC component (Accession Nos.NP_(—)607065.1; NC_(—)003485), about 45% identity from amino acids 1-657with a protein from Lactococcus lactis subsp. lactis that is afructose-specific PTS system enzyme IIBC component (EC 2.7.1.69)(Accession Nos. NP_(—)267115.1; NC_(—)002662), and 43% identity fromamino acids 1-660 with a protein from Bacillus halodurans that is a PTSsystem, fructose-specific enzyme IIBC component (Accession Nos.NP_(—)241694.1; NC_(—)002570).

A Gapped BlastP sequence alignment showed that SEQ ID NO:38 (334 aminoacids) has about 48% identity from amino acids 4-334 with a protein fromStreptococcus pneumoniae that is a sucrose operon repressor (Scr operonregulatory protein) (Accession Nos. NP_(—)359213.1; NC_(—)003098), about46% identity from amino acids 4-334 with a protein from Streptococcuspneumoniae that is a sugar-binding transcriptional regulator in the Ladfamily (Accession Nos. NP_(—)346232.1; NC_(—)003028), about 35% identityfrom amino acids 13-332 with a protein from Pediococcus pentosaceus thatis a sucrose operon repressor (Scr operon regulatory protein) (AccessionNo. sp|P43472|SCRR_PEDPE), about 35% identity from amino acids 10-334with a protein from Bacillus halodurans that is a transcriptionalrepressor of the ribose operon (Accession Nos. NP_(—)244594.1;NC_(—)002570), and 35% identity from amino acids 10-332 with a proteinfrom Streptococcus pneumoniae that is a sucrose operon repressor(Accession Nos. NP_(—)346162.1; NC_(—)003028).

A Gapped BlastP sequence alignment showed that SEQ ID NO:40 (415 aminoacids) has about 50% identity from amino acids 3-415 with a protein fromStreptococcus pneumoniae that is an ABC transporter substrate-bindingprotein (Accession Nos. NP_(—)359212.1; NC_(—)003098), about 27%identity from amino acids 19-389 with a protein from Agrobacteriumtumefaciens that is a sugar binding protein (Accession Nos.NP_(—)535638.1; NC_(—)003306), about 25% identity from amino acids11-396 with a protein from Nostoc sp. PCC 7120 that is an ABCtransporter sugar binding protein (Accession Nos. NP_(—)488317.1;NC_(—)003272), about 26% identity from amino acids 76-353 with a proteinfrom Streptomyces coelicolor that is homologous to a sugar transportsugar binding protein (Accession Nos. emb|CAB95275.1; AL359779), and 26%identity from amino acids 1-324 with a protein from Listeria innocuathat is homologous to a sugar ABC transporter, periplasmic sugar-bindingprotein (Accession Nos. NP_(—)470104.1; NC_(—)003212).

A Gapped BlastP sequence alignment showed that SEQ ID NO:42 (294 aminoacids) has about 56% identity from amino acids 10-285 with a proteinfrom Streptococcus pneumoniae that is an ABC transportermembrane-spanning permease—sugar transporter (Accession Nos.NP_(—)359211.1; NC_(—)003098), about 38% identity from amino acids 7-285with a protein from Listeria monocytogenes that is homologous to a sugarABC transporter permease protein (Accession Nos. NP_(—)464293.1;NC_(—)003210), about 38% identity from amino acids 7-285 with a proteinfrom Listeria innocua that is homologous to a sugar ABC transporterpermease protein (Accession Nos. NP_(—)470102.1; NC_(—)003212), about36% identity from amino acids 12-286 with a protein from Synechocystissp. PCC 6803 that is a lactose transport system permease protein (LacF)(Accession Nos. NP_(—)440703.1; NC_(—)000911), and 36% identity fromamino acids 11-281 with a protein from Xylella fastidiosa that is a ABCtransporter sugar permease (Accession Nos. NP_(—)299726.1;NC_(—)002488).

A Gapped BlastP sequence alignment showed that SEQ ID NO:44 (285 aminoacids) has about 59% identity from amino acids 12-285 with a proteinfrom Streptococcus pneumoniae that is an ABC transportermembrane-spanning permease—sugar transport protein (Accession Nos.NP_(—)359210.1; NC_(—)003098), about 32% identity from amino acids30-281 with a protein from Agrobacterium tumefaciens (Accession Nos.NP_(—)356672.1; NC_(—)003063), about 32% identity from amino acids30-281 with a protein from Agrobacterium tumefaciens that is an ABCtransporter, membrane spanning protein [sugar] (Accession Nos.NP_(—)534455.1; NC_(—)003305), about 33% identity from amino acids10-281 with a protein from Listeria monocytogenes that is homologous toa sugar ABC transporter, permease protein (Accession Nos.NP_(—)463711.1; NC_(—)003210), and 34% identity from amino acids 13-281with a protein from Listeria innocua that is homologous to a sugar ABCtransporter, permease protein (Accession Nos. NP_(—)469564.1;NC_(—)003212).

A Gapped BlastP sequence alignment showed that SEQ ID NO:46 (430 aminoacids) has about 36% identity from amino acids 2-429 with a protein fromStreptococcus pneumoniae that is a sucrose-6-phosphate hydrolase(Accession Nos. NP_(—)359209.1; NC_(—)003098), about 36% identity fromamino acids 2-429 with a protein from Streptococcus pneumoniae that ishomologous to a sucrose-6-phosphate hydrolase (Accession Nos.NP_(—)346228.1; NC_(—)003028), about 36% identity from amino acids18-373 with a protein from Thermotoga maritima that is abeta-fructosidase (Accession Nos. NP_(—)229215.1; NC_(—)000853), about31% identity from amino acids 21-405 with a protein from Zymomonasmobilis that is a beta-fructofuranosidase (EC 3.2.1.26) (Accession No.pir∥JU0460), and 35% identity from amino acids 21-362 with a proteinfrom Escherichia coli that is a sucrose-6 phosphate hydrolase (AccessionNos. NP_(—)311270.1; NC_(—)002695).

A Gapped BlastP sequence alignment showed that SEQ ID NO:48 (368 aminoacids) has about 65% identity from amino acids 1-366 with a protein fromStreptococcus mutans that is a multiple sugar-binding transportATP-binding protein (msmK) (Accession No. sp|Q00752|MSMK_STRMU), about65% identity from amino acids 1-366 with a protein from Streptococcuspyogenes that is a multiple sugar-binding ABC transport system(ATP-binding protein) (Accession Nos. NP_(—)269942.1; NC_(—)002737),about 66% identity from amino acids 1-367 with a protein fromStreptococcus pneumoniae that is an ABC transporter ATP-bindingprotein—multiple sugar transport (Accession Nos. NP_(—)359030.1;NC_(—)003098), about 65% identity from amino acids 1-366 with a proteinfrom Streptococcus pyogenes that is a multiple sugar-binding ABCtransport system (ATP-binding protein) (Accession Nos. NP_(—)608016.1;NC_(—)003485), and 66% identity from amino acids 1-367 with a proteinfrom Streptococcus pneumoniae that is a sugar ABC transporter,ATP-binding protein (Accession Nos. NP_(—)346026.1; NC_(—)003028).

A Gapped BlastP sequence alignment showed that SEQ ID NO:50 (490 aminoacids) has about 63% identity from amino acids 11-489 with a proteinfrom Streptococcus mutans that is a gtfA protein (Accession No.pir∥BWSOGM), about 63% identity from amino acids 11-490 with a proteinfrom Streptococcus mutans that is a sucrose phosphorylase (EC 2.4.1.7)(Accession No. pir∥A27626), about 63% identity from amino acids 11-489with a protein from Streptococcus mutans that is a sucrose phosphorylase(sucrose glucosyltransferase) (Accession No. sp|P10249|SUCP_STRMU),about 63% identity from amino acids 11-484 with a protein fromStreptococcus pneumoniae that is a dextransucrase (sucrose6-glucosyltransferase) (Accession Nos. NP_(—)359301.1; NC_(—)003098),and 63% identity from amino acids 11-484 with a protein fromStreptococcus pneumoniae that is a sucrose phosphorylase (Accession Nos.NP_(—)346325.1; NC_(—)003028).

A Gapped BlastP sequence alignment showed that SEQ ID NO:52 (328 aminoacids) has about 55% identity from amino acids 47-316 with a proteinfrom Bacillus subtilis that is a ribose ABC transporter (ribose-bindingprotein) (Accession Nos. NP_(—)391477.1; NC_(—)000964), about 45%identity from amino acids 5-323 with a protein from Lactococcus lactissubsp. lactis that is a ribose ABC transporter substrate binding protein(Accession Nos. NP_(—)267791.1; NC_(—)002662), about 42% identity fromamino acids 4-278 with a protein from Tetragenococcus halophilus that isa ribose binding protein (Accession Nos. dbj|BAA31869.1; AB009593),about 39% identity from amino acids 15-316 with a protein from Bacillushalodurans that is a ribose ABC transporter (ribose-binding protein)(Accession Nos. NP_(—)244599.1; NC_(—)002570), and 42% identity fromamino acids 4-315 with a protein from Pasteurella multocida that is anRbsB protein (Accession Nos. NP_(—)245090.1; NC_(—)002663).

A Gapped BlastP sequence alignment showed that SEQ ID NO:54 (285 aminoacids) has about 60% identity from amino acids 1-277 with a protein fromBacillus subtilis that is a ribose ABC transporter (permease) (AccessionNos. NP_(—)391476.1; NC_(—)000964), about 59% identity from amino acids1-277 with a protein from Bacillus subtilis that is a ribose transportsystem permease protein (rbcS) (Accession No. sp|P36948|RBSC_BACSU),about 57% identity from amino acids 4-277 with a protein from Bacillushalodurans that is a ribose ABC transporter (permease) (Accession Nos.NP_(—)244598.1; NC_(—)002570), about 58% identity from amino acids 4-277with a protein from Lactococcus lactis subsp. lactis that is a riboseABC transporter permease protein (Accession Nos. NP_(—)267792.1;NC_(—)002662), and 54% identity from amino acids 4-278 with a proteinfrom Haemophilus influenzae that is a D-ribose ABC transporter, permeaseprotein (rbsC) (Accession Nos. NP_(—)438661.1; NC_(—)000907).

A Gapped BlastP sequence alignment showed that SEQ ID NO:56 (496 aminoacids) has about 59% identity from amino acids 5-496 with a protein fromLactococcus lactis subsp. lactis that is a ribose ABC transporter ATPbinding protein (Accession Nos. NP_(—)267793.1; NC_(—)002662), about 57%identity from amino acids 5-496 with a protein from Bacillus subtilisthat is a ribose ABC transporter (ATP-binding protein) (Accession Nos.NP_(—)391475.1; NC_(—)000964), about 51% identity from amino acids 5-496with a protein from Bacillus subtilis that is an ATP binding protein(Accession No. pir∥I40465), about 49% identity from amino acids 5-495with a protein from Bacillus halodurans that is a ribose ABC transporter(ATP-binding protein) (Accession Nos. NP_(—)244597.1; NC_(—)002570), and45% identity from amino acids 7-494 with a protein from Agrobacteriumtumefaciens that is an ABC transporter, nucleotide binding/ATPaseprotein [ribose] (Accession Nos. NP_(—)533484.1; NC_(—)003304).

A Gapped BlastP sequence alignment showed that SEQ ID NO:58 (134 aminoacids) has about 58% identity from amino acids 4-134 with a protein fromLactobacillus sakei that is a ribose permease (RbsD) (Accession Nos.gb|AAD34337.1; AF115391), about 51% identity from amino acids 4-134 witha protein from Clostridium perfringens that is homologous to a riboseABC transporter (Accession Nos. NP_(—)562547.1; NC_(—)003366), about 50%identity from amino acids 4-132 with a protein from Lactococcus lactissubsp. lactis that is a ribose ABC transporter permease protein(Accession Nos. NP_(—)267794.1; NC_(—)002662), about 45% identity fromamino acids 4-134 with a protein from Bacillus halodurans that is aribose ABC transporter (permease) (Accession Nos. NP_(—)244596.1;NC_(—)002570), and 51% identity from amino acids 4-134 with a proteinfrom Staphylococcus aureus subsp. aureus that is a ribose permease(Accession Nos. NP_(—)370793.1; NC_(—)002758).

A Gapped BlastP sequence alignment showed that SEQ ID NO:60 (308 aminoacids) has about 51% identity from amino acids 4-301 with a protein fromLactobacillus sakei that is a ribokinase (RbsK) (Accession Nos.gb|AAD34338.1; AF115391), about 48% identity from amino acids 1-303 witha protein from Staphylococcus aureus subsp. aureus that is homologous toa ribokinase (Accession Nos. NP_(—)370792.1; NC_(—)002758), about 45%identity from amino acids 3-305 with a protein from Clostridiumperfringens that is a ribokinase (Accession Nos. NP_(—)562548.1;NC_(—)003366), about 41% identity from amino acids 1-299 with a proteinfrom Haemophilus influenzae that is a ribokinase (RbsK) (Accession Nos.NP_(—)438663.1; NC_(—)000907), and 38% identity from amino acids 2-300with a protein from Yersinia pestis that is a ribokinase (Accession Nos.NP_(—)403674.1; NC_(—)003143).

A Gapped BlastP sequence alignment showed that SEQ ID NO:62 (285 aminoacids) has about 63% identity from amino acids 1-285 with a protein fromLactococcus lactis subsp. lactis that is a maltose ABC transporterpermease protein (Accession Nos. NP_(—)267841.1; NC_(—)002662), about54% identity from amino acids 6-284 with a protein from Streptococcuspyogenes that is homologous to a maltose/maltodextrin ABC transportsystem protein (permease) (Accession Nos. NP_(—)269423.1; NC_(—)002737),about 38% identity from amino acids 12-284 with a protein fromKlebsiella oxytoca that is homologous to a maIG protein (Accession No.pir∥S63616), about 39% identity from amino acids 9-285 with a proteinfrom Bacillus halodurans that is a maltose/maltodextrin transport system(permease) (Accession Nos. NP_(—)243790.1; NC_(—)002570), and 36%identity from amino acids 7-285 with a protein from Bacillus subtilisthat is homologous to a maltodextrin transport system permease(Accession Nos. NP_(—)391294.1; NC_(—)000964).

A Gapped BlastP sequence alignment showed that SEQ ID NO:64 (452 aminoacids) has about 63% identity from amino acids 1-452 with a protein fromLactococcus lactis subsp. lactis that is a maltose ABC transporterpermease protein (Accession Nos. NP_(—)267840.1; NC_(—)002662), about52% identity from amino acids 3-452 with a protein from Streptococcuspyogenes that is homologous to a maltose/maltodextrin ABC transportsystem protein (permease) (Accession Nos. NP_(—)269422.1; NC_(—)002737),about 52% identity from amino acids 3-452 with a protein fromStreptococcus pyogenes that is homologous to a maltose/maltodextrin ABCtransport system (permease) (Accession Nos. NP_(—)607422.1;NC_(—)003485), about 34% identity from amino acids 28-451 with a proteinfrom Klebsiella oxytoca that is homologous to a malF protein (AccessionNo. pir∥S63615), and 33% identity from amino acids 23-451 with a proteinfrom Bacillus halodurans that is a maltose/maltodextrin transport systempermease (Accession Nos. NP_(—)243791.1; NC_(—)002570).

A Gapped BlastP sequence alignment showed that SEQ ID NO:66 (408 aminoacids) has about 49% identity from amino acids 1-407 with a protein fromLactococcus lactis subsp. lactis that is a maltose ABC transportersubstrate binding protein (Accession Nos. NP_(—)267839.1; NC_(—)002662),about 37% identity from amino acids 1-405 with a protein fromStreptococcus pyogenes that is homologous to amaltose/maltodextrin-binding protein (Accession Nos. NP_(—)607421.1;NC_(—)003485), about 36% identity from amino acids 1-405 with a proteinfrom Streptococcus pyogenes that is homologous to amaltose/maltodextrin-binding protein (Accession Nos. NP_(—)269421.1;NC_(—)002737), about 27% identity from amino acids 1-393 with a proteinfrom Listeria innocua that is homologous to a maltose/maltodextrinABC-transporter (binding protein) (Accession Nos. NP_(—)471563.1;NC_(—)003212), and 26% identity from amino acids 1-403 with a proteinfrom Bacillus subtilis that is homologous to amaltose/maltodextrin-binding protein (Accession Nos. NP_(—)391296.1;NC_(—)000964).

A Gapped BlastP sequence alignment showed that SEQ ID NO:68 (368 aminoacids) has about 64% identity from amino acids 1-366 with a protein fromStreptococcus mutans that is a multiple sugar-binding transportATP-binding protein (msmK) (Accession No. sp|Q00752|MSMK_STRMU), about64% identity from amino acids 1-366 with a protein from Streptococcuspyogenes that is a multiple sugar-binding ABC transport system(ATP-binding) protein (Accession Nos. NP_(—)269942.1; NC_(—)002737),about 64% identity from amino acids 1-366 with a protein fromStreptococcus pyogenes that is a multiple sugar-binding ABC transportsystem (ATP-binding) protein (Accession Nos. NP_(—)608016.1;NC_(—)003485), about 64% identity from amino acids 1-366 with a proteinfrom Streptococcus pneumoniae that is an ABC transporter ATP-bindingprotein—multiple sugar transport (Accession Nos. NP_(—)359030.1;NC_(—)003098), and 62% identity from amino acids 1-368 with a proteinfrom Lactococcus lactis subsp. lactis that is a multiple sugar ABCtransporter ATP-binding protein (Accession Nos. NP_(—)266577.1;NC_(—)002662).

A Gapped BlastP sequence alignment showed that SEQ ID NO:70 (512 aminoacids) has about 60% identity from amino acids 1-510 with a protein fromStreptococcus pyogenes that is homologous to a sugar ABC transporter(ATP-binding protein) (Accession Nos. NP_(—)269365.1; NC_(—)002737),about 60% identity from amino acids 1-510 with a protein fromStreptococcus pyogenes that is homologous to a sugar ABC transporter(ATP-binding protein) (Accession Nos. NP_(—)607296.1; NC_(—)003485),about 59% identity from amino acids 5-503 with a protein fromLactococcus lactis subsp. lactis that is a sugar ABC transporter ATPbinding protein (Accession Nos. NP_(—)267484.1; NC_(—)002662), about 61%identity from amino acids 7-503 with a protein from Streptococcuspneumoniae that is a sugar ABC transporter, ATP-binding protein(Accession Nos. NP_(—)345337.1; NC_(—)003028), and 60% identity fromamino acids 7-503 with a protein from Streptococcus pneumoniae that is aABC transporter ATP-binding protein—ribose/galactose transport(Accession Nos. NP_(—)358342.1; NC_(—)003098).

A Gapped BlastP sequence alignment showed that SEQ ID NO:72 (383 aminoacids) has about 49% identity from amino acids 7-351 with a protein fromLactococcus lactis subsp. lactis that is a sugar ABC transporterpermease protein (Accession Nos. NP_(—)267485.1; NC_(—)002662), about47% identity from amino acids 4-351 with a protein from Streptococcuspneumoniae that is an ABC transporter membrane-spanning permease(ribose/galactose transport) (Accession Nos. NP_(—)358343.1;NC_(—)003098), about 47% identity from amino acids 4-351 with a proteinfrom Streptococcus pneumoniae that is homologous to a sugar ABCtransporter, permease protein (Accession Nos. NP_(—)345338.1;NC_(—)003028), about 49% identity from amino acids 4-342 with a proteinfrom Streptococcus pyogenes that is homologous to a sugar ABCtransporter (permease protein) (Accession Nos. NP_(—)269364.1;NC_(—)002737), and 49% identity from amino acids 4-342 with a proteinfrom Streptococcus pyogenes that is homologous to a sugar ABCtransporter (permease protein) (Accession Nos. NP_(—)607295.1;NC_(—)003485).

A Gapped BlastP sequence alignment showed that SEQ ID NO:74 (318 aminoacids) has about 67% identity from amino acids 1-318 with a protein fromStreptococcus pyogenes that is homologous to a sugar ABC transporter(permease protein) (Accession Nos. NP_(—)607294.1; NC_(—)003485), about66% identity from amino acids 1-318 with a protein from Streptococcuspyogenes that is homologous to a sugar ABC transporter (permeaseprotein) (Accession Nos. NP_(—)269363.1; NC_(—)002737), about 65%identity from amino acids 1-318 with a protein from Streptococcuspneumoniae that is homologous to a sugar ABC transporter, permeaseprotein (Accession Nos. NP_(—)345339.1; NC_(—)003028), about 63%identity from amino acids 1-318 with a protein from Lactococcus lactissubsp. lactis that is a sugar ABC transporter permease protein(Accession Nos. NP_(—)267486.1; NC_(—)002662), and 61% identity fromamino acids 6-318 with a protein from Listeria innocua that ishomologous to a sugar ABC transporter (permease protein) (Accession Nos.NP_(—)470764.1; NC_(—)003212).

A Gapped BlastP sequence alignment showed that SEQ ID NO:76 (450 aminoacids) has about 68% identity from amino acids 11-448 with a proteinfrom Neisseria meningitidis that is homologous to a sugar transporter(Accession Nos. NP_(—)273437.1; NC_(—)003112), about 68% identity fromamino acids 11-448 with a protein from Neisseria meningitidis that ishomologous to an integral membrane transport protein (Accession Nos.NP_(—)284797.1; NC_(—)003116), about 39% identity from amino acids17-229 with a protein from Caulobacter crescentus that is homologous toa transporter (Accession Nos. NP_(—)421086.1; NC_(—)002696), about 21%identity from amino acids 31-450 with a protein from Lycopersiconesculentum that is a sucrose transporter (Accession Nos. gb|AAG09270.1;AF176950), and 21% identity from amino acids 31-442 with a protein fromArabidopsis thaliana that is a sucrose transporter (Accession Nos.gb|AAG09191.1; AF175321).

A Gapped BlastP sequence alignment showed that SEQ ID NO:78 (495 aminoacids) has about 32% identity from amino acids 8-482 with a protein fromLactococcus lactis subsp. lactis that is a transporter protein(Accession Nos. NP_(—)266394.1; NC_(—)002662), about 34% identity fromamino acids 8-482 with a protein from Listeria monocytogenes that ishomologous to an efflux transporter (Accession Nos. NP_(—)464506.1;NC_(—)003210), about 34% identity from amino acids 8-482 with a proteinfrom Listeria innocua that is homologous to an efflux transporter(Accession Nos. NP_(—)470317.1; NC_(—)003212), about 30% identity fromamino acids 7-422 with a protein from Clostridium acetobutylicum that isan MDR related permease (Accession Nos. NP_(—)149294.1; NC_(—)001988),and 29% identity from amino acids 8-425 with a protein from Streptomycescoelicolor that is homologous to a membrane transport protein (AccessionNos. emb|CAB89031.1; AL353870).

A Gapped BlastP sequence alignment showed that SEQ ID NO:80 (471 aminoacids) has about 32% identity from amino acids 1-440 with a protein fromLactococcus lactis subsp. lactis that is a transporter protein(Accession Nos. NP_(—)266394.1; NC_(—)002662), about 34% identity fromamino acids 1-464 with a protein from Listeria monocytogenes that ishomologous to an efflux transporter (Accession Nos. NP_(—)464506.1;NC_(—)003210), about 34% identity from amino acids 1-464 with a proteinfrom Listeria innocua that is homologous to an efflux transporter(Accession Nos. NP_(—)470317.1; NC_(—)003212), about 29% identity fromamino acids 1-412 with a protein from Clostridium acetobutylicum that isan MDR related permease (Accession Nos. NP_(—)149294.1; NC_(—)001988),and 28% identity from amino acids 4-459 with a protein from Streptomycescoelicolor that is homologous to an exporter (Accession No. pir∥T36377).

A Gapped BlastP sequence alignment showed that SEQ ID NO:82 (412 aminoacids) has about 49% identity from amino acids 18-400 with a proteinfrom Listeria innocua that is homologous to a drug-efflux transporter(Accession Nos. NP_(—)472212.1; NC_(—)003212), about 49% identity fromamino acids 18-400 with a protein from Listeria monocytogenes that ishomologous to a drug-efflux transporter (Accession Nos. NP_(—)466263.1;NC_(—)003210), about 48% identity from amino acids 18-397 with a proteinfrom Escherichia coli that is homologous to a transport protein(Accession Nos. NP_(—)415571.1; NC_(—)000913), about 47% identity fromamino acids 15-399 with a protein from Lactococcus lactis subsp. lactisthat is a multidrug resistance efflux pump (Accession Nos.NP_(—)266282.1; NC_(—)002662), and 48% identity from amino acids 18-399with a protein from Salmonella typhimurium that is homologous to an MFSfamily transport protein (Accession Nos. NP_(—)460125.1; NC_(—)003197).

A Gapped BlastP sequence alignment showed that SEQ ID NO:84 (462 aminoacids) has about 38% identity from amino acids 9-413 with ORFC fromOenococcus oeni (Accession Nos. emb|CAB61253.1; AJ250422), about 38%identity from amino acids 2-378 with a protein from Lactococcus lactissubsp. lactis that is a transporter protein (Accession Nos.NP_(—)267695.1; NC_(—)002662), about 34% identity from amino acids 6-411with a protein from Streptococcus pyogenes that is homologous to a drugresistance protein (Accession Nos. NP_(—)606824.1; NC_(—)003485), about33% identity from amino acids 6-411 with a protein from Streptococcuspyogenes that is homologous to a drug resistance protein (Accession Nos.NP_(—)268834.1; NC_(—)002737), and 34% identity from amino acids 2-454with a protein from Lactococcus lactis subsp. lactis that is adrug-export protein (Accession Nos. NP_(—)267504.1; NC_(—)002662).

A Gapped BlastP sequence alignment showed that SEQ ID NO:86 (490 aminoacids) has about 55% identity from amino acids 3-476 with a protein fromListeria monocytogenes that is homologous to a drug-export protein(Accession Nos. NP_(—)466111.1; NC_(—)003210), about 54% identity fromamino acids 3-476 with a protein from Listeria innocua that ishomologous to a drug-export protein (Accession Nos. NP_(—)472062.1;NC_(—)003212), about 45% identity from amino acids 6-478 with a proteinfrom Lactococcus lactis subsp. lactis that is a multidrug resistanceprotein (Accession Nos. NP_(—)267065.1; NC_(—)002662), about 49%identity from amino acids 8-484 with a protein from Bacillus subtilisthat is homologous to a multidrug resistance protein (Accession Nos.NP_(—)388266.1; NC_(—)000964), and 44% identity from amino acids 18-425with a protein from Bacillus subtilis that is homologous to a multidrugresistance protein (Accession Nos. NP_(—)388782.1; NC_(—)000964).

A Gapped BlastP sequence alignment showed that SEQ ID NO:88 (416 aminoacids) has about 26% identity from amino acids 17-408 with a proteinfrom Desulfitobacterium hafniense (Accession Nos. gb|AAL87781.1;AF403184), about 25% identity from amino acids 26-408 with a proteinfrom Streptococcus pneumoniae that is transporter in the majorfacilitator superfamily (Accession Nos. NP_(—)359046.1; NC_(—)003098),about 21% identity from amino acids 61-399 with a protein fromCampylobacter jejuni that is homologous to an efflux protein (AccessionNos. NP_(—)282813.1; NC_(—)002163), about 19% identity from amino acids25-368 with a protein from Agrobacterium tumefaciens that is homologousto an MFS permease (Accession Nos. NP_(—)533033.1; NC_(—)003304), and25% identity from amino acids 19-205 with a protein from Bacillushalodurans that is a multidrug resistance protein (Accession Nos.NP_(—)244175.1; NC_(—)002570).

A Gapped BlastP sequence alignment showed that SEQ ID NO:90 (548 aminoacids) has about 38% identity from amino acids 17-546 with a proteinfrom Listeria innocua that is homologous to a transporter protein(Accession Nos. NP_(—)471001.1; NC_(—)003212), about 37% identity fromamino acids 17-546 with a protein from Listeria monocytogenes that ishomologous to a transporter protein (Accession Nos. NP_(—)465149.1;NC_(—)003210), about 36% identity from amino acids 1-534 with a proteinfrom Streptococcus pneumoniae that is a polysaccharide transporter(Accession Nos. NP_(—)358976.1; NC_(—)003098), about 36% identity fromamino acids 17-534 with a protein from Streptococcus pneumoniae that ishomologous to a polysaccharide biosynthesis protein (Accession Nos.NP_(—)345978.1; NC_(—)003028), and 35% identity from amino acids 12-546with a hypothetical protein from Lactococcus lactis subsp. lactis(Accession Nos. NP_(—)267962.1; NC_(—)002662).

A Gapped BlastP sequence alignment showed that SEQ ID NO:92 (485 aminoacids) has about 44% identity from amino acids 1-484 with a protein fromListeria monocytogenes that is homologous to an efflux transporterprotein (Accession Nos. NP_(—)464506.1; NC_(—)003210), about 44%identity from amino acids 1-484 with a protein from Listeria innocuathat is homologous to an efflux transporter protein (Accession Nos.NP_(—)470317.1; NC_(—)003212), about 34% identity from amino acids 9-420with a protein from Clostridium acetobutylicum that is an MDR-relatedpermease (Accession Nos. NP_(—)149294.1; NC_(—)001988), about 33%identity from amino acids 12-475 with a protein from Lactococcus lactissubsp. lactis that is a transporter protein (Accession Nos.NP_(—)266394.1; NC_(—)002662), and 34% identity from amino acids 1-457with a hypothetical protein from Myxococcus xanthus (Accession Nos.emb|CAB37973.1; X76640).

A Gapped BlastP sequence alignment showed that SEQ ID NO:94 (199 aminoacids) has about 46% identity from amino acids 23-173 with a proteinfrom Listeria innocua that is homologous to a drug-efflux transporterprotein (Accession Nos. NP_(—)472212.1; NC_(—)003212), about 45%identity from amino acids 23-173 with a protein from Listeriamonocytogenes that is homologous to a drug-efflux transporter protein(Accession Nos. NP_(—)466263.1; NC_(—)003210), about 49% identity fromamino acids 23-173 with a protein from Lactococcus lactis subsp. lactisthat is a multidrug resistance efflux pump (Accession Nos.NP_(—)266282.1; NC_(—)002662), about 46% identity from amino acids23-173 with a protein from Salmonella enterica subsp. enterica serovarTyphi that is homologous to an efflux pump (Accession Nos.NP_(—)454977.1; NC_(—)003198), and 46% identity from amino acids 23-173with a protein from Salmonella typhimurium that is homologous to apermease (Accession Nos. NP_(—)459377.1; NC_(—)003197).

A Gapped BlastP sequence alignment showed that SEQ ID NO:96 (538 aminoacids) has about 32% identity from amino acids 4-525 with a protein fromStreptococcus pneumoniae that is a polysaccharide transporter (AccessionNos. NP_(—)358976.1; NC_(—)003098), about 32% identity from amino acids5-525 with a protein from Streptococcus pneumoniae that is homologous toa polysaccharide biosynthesis protein (Accession Nos. NP_(—)345978.1;NC_(—)003028), about 33% identity from amino acids 5-526 with aconserved hypothetical protein from Streptococcus pyogenes (AccessionNos. NP_(—)606680.1; NC_(—)003485), about 33% identity from amino acids5-526 with a conserved hypothetical protein from Streptococcus pyogenes(Accession Nos. NP_(—)268708.1; NC_(—)002737), and 30% identity fromamino acids 4-526 with a hypothetical protein from Lactococcus lactissubsp. lactis (Accession Nos. NP_(—)267962.1; NC_(—)002662).

A Gapped BlastP sequence alignment showed that SEQ ID NO:98 (328 aminoacids) has about 57% identity from amino acids 1-323 with a protein fromPediococcus pentosaceus that is a sucrose operon regulatory protein(scrR) (Accession No. sp|P43472|SCRR_PEDPE), about 51% identity fromamino acids 1-322 with a protein from Streptococcus pneumoniae that is asucrose operon repressor (Accession Nos. NP_(—)346162.1; NC_(—)003028),about 49% identity from amino acids 1-326 with a protein fromStreptococcus mutans that is a sucrose operon regulatory protein (scrR)(Accession No. sp|54430|SCRR_STRMU), about 49% identity from amino acids1-322 with a protein from Streptococcus pyogenes that is homologous to asucrose operon repressor (Accession Nos. NP_(—)607889.1; NC_(—)003485),and 49% identity from amino acids 1-322 with a protein fromStreptococcus pyogenes that is homologous to a sucrose operon repressor(Accession Nos. NP_(—)269821.1; NC_(—)002737).

A Gapped BlastP sequence alignment showed that SEQ ID NO:100 (485 aminoacids) has about 50% identity from amino acids 1-466 with a protein fromStreptococcus sobrinus that is a sucrose-6-phosphate hydrolase (ScrB)(Accession No. pir∥S68598), about 49% identity from amino acids 1-461with a protein from Streptococcus pneumoniae that is asucrose-6-phosphate hydrolase (Accession Nos. NP_(—)359160.1;NC_(—)003098), about 49% identity from amino acids 1-461 with a proteinfrom Streptococcus pneumoniae that is a sucrose-6-phosphate hydrolase(Accession Nos. NP_(—)346161.1; NC_(—)003028), about 49% identity fromamino acids 1-466 with a protein from Streptococcus pyogenes that ishomologous to a sucrose-6-phosphate hydrolase (Accession Nos.NP_(—)607888.1; NC_(—)003485), and 49% identity from amino acids 1-466with a protein from Streptococcus pyogenes that is homologous to asucrose-6-phosphate hydrolase (Accession Nos. NP_(—)269820.1;NC_(—)002737).

A Gapped BlastP sequence alignment showed that SEQ ID NO:102 (649 aminoacids) has about 65% identity from amino acids 1-645 with a protein fromStreptococcus mutans that is a phosphotransferase system enzyme II (EC2.7.1.69), sucrose-specific IIABC component (Accession No.sp|P12655|PTSA_STRMU), about 56% identity from amino acids 1-647 with aprotein from Pediococcus pentosaceus that is a phosphotransferase systemenzyme II (EC 2.7.1.69), sucrose specific enzyme IIABC (Accession No.sp|P43470|PTSA_PEDPE), about 52% identity from amino acids 1-643 with aprotein from Lactococcus lactis that is an enzyme II sucrose protein(Accession Nos. emb|CAB09690.1; Z97015), about 52% identity from aminoacids 114-647 with a protein from Lactobacillus sakei that is asucrose-specific enzyme II of the PTS (Accession Nos. gb|AAK92528.1;AF401046), and 45% identity from amino acids 1-621 with a protein fromCorynebacterium glutamicum that is a phosphotransferase system IIBcomponent (Accession Nos. NP_(—)601842.1; NC_(—)003450).

A Gapped BlastP sequence alignment showed that SEQ ID NO:104 (667 aminoacids) has about 42% identity from amino acids 192-661 with a proteinfrom Lactococcus lactis subsp. lactis that is a beta-glucoside-specificPTS system IIABC component (EC 2.7.1.69) (Accession Nos. NP_(—)266583.1;NC_(—)002662), about 39% identity from amino acids 191-652 with aprotein from Listeria monocytogenes that is homologous to aphosphotransferase system (PTS) beta-glucoside-specific enzyme IIABC(Accession Nos. NP_(—)464560.1; NC_(—)003210), about 37% identity fromamino acids 191-662 with a protein from Clostridium longisporum that isa PTS-dependent enzyme II (Accession Nos. gb|AACO5713.1; L49336), about36% identity from amino acids 191-666 with a protein from Bacillushalodurans that is a PTS system, beta-glucoside-specific enzyme II, ABCcomponent (Accession Nos. NP_(—)241461.1; NC_(—)002570), and 36%identity from amino acids 191-650 with a protein from Listeria innocuathat is homologous to a PTS system, beta-glucosides specific enzymeIIABC (Accession Nos. NP_(—)469373.1; NC_(—)003212).

A Gapped BlastP sequence alignment showed that SEQ ID NO:106 (241 aminoacids) has about 47% identity from amino acids 1-238 with a protein fromBacillus subtilis that is a trehalose operon transcriptional repressor(Accession No. sp|P39796|TRER_BACSU), about 41% identity from aminoacids 4-238 with a protein from Bacillus halodurans that is atranscriptional repressor of the trehalose operon (Accession Nos.NP_(—)241739.1; NC_(—)002570), about 44% identity from amino acids 9-237with a protein from Listeria innocua that is homologous to atranscription regulator GntR family (Accession Nos. NP_(—)470558.1;NC_(—)003212), about 44% identity from amino acids 9-237 with a proteinfrom Listeria monocytogenes that is homologous to a transcriptionregulator GntR family (Accession Nos. NP_(—)464778.1; NC_(—)003210), and41% identity from amino acids 5-238 with a protein from Lactococcuslactis subsp. lactis that is a GntR family transcriptional regulator(Accession Nos. NP_(—)266581.1; NC_(—)002662).

A Gapped BlastP sequence alignment showed that SEQ ID NO:108 (570 aminoacids) has about 56% identity from amino acids 22-566 with a proteinfrom Streptococcus pyogenes that is homologous to a dextran glucosidase(Accession Nos. NP_(—)608103.1; NC_(—)003485), about 57% identity fromamino acids 23-568 with a protein from Streptococcus pneumoniae that isa dextran glucosidase (Accession Nos. NP_(—)359290.1; NC_(—)003098),about 56% identity from amino acids 22-566 with a protein fromStreptococcus pyogenes that is homologous to a dextran glucosidase(Accession Nos. NP_(—)270026.1; NC_(—)002737), about 57% identity fromamino acids 23-568 with a protein from Streptococcus pneumoniae that ishomologous to a dextran glucosidase DexS (Accession Nos. NP_(—)346315.1;NC_(—)003028), and 54% identity from amino acids 17-570 with a proteinfrom Clostridium perfringens that is an alpha-glucosidase (AccessionNos. NP_(—)561478.1; NC_(—)003366).

A Gapped BlastP sequence alignment showed that SEQ ID NO:110 (370 aminoacids) has about 67% identity from amino acids 1-368 with a protein fromStreptococcus pneumoniae that is an ABC transporter ATP-bindingprotein—multiple sugar transport (Accession Nos. NP_(—)359030.1;NC_(—)003098), about 67% identity from amino acids 1-368 with a proteinfrom Streptococcus pneumoniae that is a sugar ABC transporter,ATP-binding protein (Accession Nos. NP_(—)346026.1; NC_(—)003028), about66% identity from amino acids 1-368 with a protein from Streptococcusmutans that is a multiple sugar-binding transport ATP-binding protein(msmK) (Accession No. sp|Q00752|MSMK_STRMU), about 68% identity fromamino acids 1-365 with a protein from Listeria innocua that ishomologous to a sugar ABC transporter, ATP-binding protein (AccessionNos. NP_(—)469649.1; NC_(—)003212), and 67% identity from amino acids1-365 with a protein from Listeria monocytogenes that is homologous to asugar ABC transporter, ATP-binding protein (Accession Nos.NP_(—)463809.1; NC_(—)003210).

A Gapped BlastP sequence alignment showed that SEQ ID NO:112 (278 aminoacids) has about 81% identity from amino acids 2-278 with a protein fromStreptococcus mutans that is a multiple sugar-binding transport systempermease protein (msmG) (Accession No. sp|Q00751|MSMG_STRMU), about 73%identity from amino acids 1-278 with a protein from Streptococcuspneumoniae that is a sugar ABC transporter, permease protein (AccessionNos. NP_(—)346326.1; NC_(—)003028), about 72% identity from amino acids2-278 with a protein from Streptococcus pneumoniae that is a ABCtransporter membrane spanning permease—multiple sugars (Accession Nos.NP_(—)359302.1; NC_(—)003098), about 85% identity from amino acids72-278 with a hypothetical protein fragment from Streptococcus mutans(Accession No. pir∥B27626), and 44% identity from amino acids 4-278 witha protein from Clostridium acetobutylicum that is a sugar permease(Accession Nos. NP_(—)350251.1; NC_(—)003030).

A Gapped BlastP sequence alignment showed that SEQ ID NO:114 (291 aminoacids) has about 73% identity from amino acids 4-290 with a protein fromStreptococcus pneumoniae that is an ABC transporter membrane-spanningpermease—multiple sugars (Accession Nos. NP_(—)359303.1; NC_(—)003098),about 73% identity from amino acids 4-290 with a protein fromStreptococcus pneumoniae that is a sugar ABC transporter, permeaseprotein (Accession Nos. NP_(—)346327.1; NC_(—)003028), about 73%identity from amino acids 1-290 with a protein from Streptococcus mutansthat is a multiple sugar-binding transport system permease protein(msmF) (Accession No. sp|Q00750|MSMF_STRMU), about 53% identity fromamino acids 6-291 with a protein from Clostridium acetobutylicum that isan ABC-type sugar transport system, permease component (Accession Nos.NP_(—)350252.1; NC_(—)003030), and 32% identity from amino acids 2-291with a protein from Thermoanaerobacterium thermosulfurigenes that is apotential starch degradation products transport system permease protein(Accession No. sp|P37730|AMYD_THETU).

A Gapped BlastP sequence alignment showed that SEQ ID NO:116 (423 aminoacids) has about 60% identity from amino acids 8-421 with a protein fromStreptococcus mutans that is a multiple sugar-binding protein precursor(Accession No. sp|Q00749|MSME_STRMU), about 56% identity from aminoacids 9-421 with a protein from Streptococcus pneumoniae that is a sugarABC transporter, sugar-binding protein (Accession Nos. NP_(—)346328.1;NC_(—)003028), about 56% identity from amino acids 9-421 with a proteinfrom Streptococcus pneumoniae that is an ABC transportersubstrate-binding protein—multiple sugars (Accession Nos.NP_(—)359304.1; NC_(—)003098), about 29% identity from amino acids 9-420with a protein from Clostridium acetobutylicum that is an ABC-type sugartransport system, periplasmic sugar-binding component (Accession Nos.NP_(—)350253.1; NC_(—)003030), and 24% identity from amino acids 6-412with a protein from Bacillus subtilis that is homologous to a multiplesugar-binding protein (Accession Nos. NP_(—)391140.1; NC_(—)000964).

A Gapped BlastP sequence alignment showed that SEQ ID NO:118 (279 aminoacids) has about 57% identity from amino acids 1-273 with a protein fromPediococcus pentosaceus that is a raffinose operon transcriptionalregulatory protein (rafR) (Accession No. sp|P43465|RAFR_PEDPE), about35% identity from amino acids 5-273 with a protein from Streptococcusmutans that is homologous to a transcription regulator (msmR) (AccessionNo. pir∥A42400), about 35% identity from amino acids 5-273 with aprotein from Streptococcus mutans that is an msm operon regulatoryprotein (Accession No. sp|Q00753|MSMR_STRMU), about 36% identity fromamino acids 19-273 with a protein from Streptococcus pneumoniae that isan msm operon regulatory protein (Accession Nos. NP_(—)346330.1;NC_(—)003028), and 36% identity from amino acids 19-273 with a proteinfrom Streptococcus pneumoniae that is an msm (multiple sugar metabolism)operon regulatory protein (Accession Nos. NP_(—)359306.1; NC_(—)003098).

A Gapped BlastP sequence alignment showed that SEQ ID NO:120 (277 aminoacids) has about 28% identity from amino acids 37-141 with a proteinfrom Treponema pallidum that is homologous to an rRNA methylase(Accession Nos. NP_(—)218549.1; NC_(—)000919), about 32% identity fromamino acids 74-141 with a protein from Guillardia theta that is aGTP-binding nuclear protein RAN (Accession Nos. NP_(—)113408.1;NC_(—)002753), about 29% identity from amino acids 75-141 with a proteinfrom Dictyostelium discoideum that is a GTP-binding nuclear proteinRAN/TC4 (Accession No. sp|P33519|RAN DICDI), and about 25% identity fromamino acids 140-190 with a putative protein from Arabidopsis thaliana(Accession Nos. NP_(—)191798.1; NM_(—)116104).

A Gapped BlastP sequence alignment showed that SEQ ID NO:122 (530 aminoacids) has about 26% identity from amino acids 8-524 with a protein fromLactococcus lactis subsp. lactis that is an ABC transporter ATP bindingand permease protein (Accession Nos. NP_(—)267678.1; NC_(—)002662),about 25% identity from amino acids 49-518 with a protein fromStreptococcus pneumoniae that is an ABC transporter, ATP-binding protein(Accession Nos. NP_(—)344680.1; NC_(—)003028), about 25% identity fromamino acids 49-518 with a protein from Streptococcus pneumoniae that isan ABC transporter ATP-binding/membrane spanning permease (AccessionNos. NP_(—)357731.1; NC_(—)003098), about 24% identity from amino acids47-511 with a protein from Synechocystis sp. PCC 6803 that is an ABCtransporter (Accession Nos. NP_(—)440626.1; NC_(—)000911), and 24%identity from amino acids 7-511 with a protein from Bacillus subtilisthat is homologous to an ABC transporter (ATP-binding protein)(Accession Nos. NP_(—)388852.1; NC_(—)000964).

A Gapped BlastP sequence alignment showed that SEQ ID NO:124 (530 aminoacids) has about 24% identity from amino acids 4-524 with a protein fromLactococcus lactis subsp. lactis that is an ABC transporter ATP bindingand permease protein (Accession Nos. NP_(—)267678.1; NC_(—)002662),about 25% identity from amino acids 55-508 with a protein fromStreptococcus pneumoniae that is an ABC transporter, ATP-binding protein(Accession Nos. NP_(—)344680.1; NC_(—)003028), about 25% identity fromamino acids 55-508 with a protein from Streptococcus pneumoniae that isan ABC transporter ATP-binding/membrane spanning permease (AccessionNos. NP_(—)357731.1; NC_(—)003098), about 24% identity from amino acids1-511 with a protein from Streptococcus pneumoniae that is a drug effluxABC transporter, ATP-binding/permease (Accession Nos. NP_(—)345800.1;NC_(—)003028), and 24% identity from amino acids 1-511 with a proteinfrom Streptococcus pneumoniae that is an ABC transporterATP-binding/membrane spanning protein (Accession Nos. NP_(—)358796.1;NC_(—)003098).

A Gapped BlastP sequence alignment showed that SEQ ID NO:126 (527 aminoacids) has about 25% identity from amino acids 8-527 with a protein fromLactococcus lactis subsp. lactis that is an ABC transporter ATP bindingand permease protein (Accession Nos. NP_(—)267678.1; NC_(—)002662),about 24% identity from amino acids 13-520 with a protein fromStreptococcus pneumoniae that is an ABC transporter ATP-binding/membranespanning permease protein (Accession Nos. NP_(—)357731.1; NC_(—)003098),about 24% identity from amino acids 13-520 with a protein fromStreptococcus pneumoniae that is an ABC transporter, ATP-binding protein(Accession Nos. NP_(—)344680.1; NC_(—)003028), about 22% identity fromamino acids 22-511 with a protein from Streptococcus pneumoniae that isa drug efflux ABC transporter, ATP-binding/permease protein (AccessionNos. NP_(—)345800.1; NC_(—)003028), and 22% identity from amino acids22-511 with a protein from Streptococcus pneumoniae that is an ABCtransporter ATP-binding/membrane spanning protein (Accession Nos.NP_(—)358796.1; NC_(—)003098).

A Gapped BlastP sequence alignment showed that SEQ ID NO:128 (534 aminoacids) has about 23% identity from amino acids 14-512 with a proteinfrom Streptococcus pneumoniae that is a comA protein (Accession No.pir∥A39203), about 26% identity from amino acids 3-512 with a proteinfrom Lactococcus lactis that is a Lactococcin A transport ATP-bindingprotein (lcnC) (Accession No. sp|Q00564|LCNC_LACLA), about 23% identityfrom amino acids 14-512 with a protein from Streptococcus pneumoniaethat is a transport ATP-binding protein (ComA) (Accession Nos.NP_(—)357637.1; NC_(—)003098), about 25% identity from amino acids113-509 with a protein from Streptococcus salivarius that is an ABCtransporter (Accession Nos. gb|AAC72026.1; AF043280), and 22% identityfrom amino acids 14-512 with a protein from Streptococcus pneumoniaethat is a competence factor transporting ATP-binding/permease protein(ComA) (Accession Nos. NP_(—)344591.1; NC_(—)003028).

A Gapped BlastP sequence alignment showed that SEQ ID NO:130 (527 aminoacids) has about 23% identity from amino acids 16-524 with a proteinfrom Lactococcus lactis subsp. lactis that is an ABC transporter ATPbinding and permease protein (Accession Nos. NP_(—)267678.1;NC_(—)002662), about 25% identity from amino acids 6-520 with a proteinfrom Streptococcus pneumoniae that is an ABC transporter, ATP-bindingprotein (Accession Nos. NP_(—)344680.1; NC_(—)003028), about 25%identity from amino acids 6-520 with a protein from Streptococcuspneumoniae that is an ABC transporter ATP-binding/membrane spanningpermease (Accession Nos. NP_(—)357731.1; NC_(—)003098), about 24%identity from amino acids 105-511 with a protein from Streptococcuspneumoniae that is an ABC transporter ATP-binding/membrane spanningprotein (Accession Nos. NP_(—)358796.1; NC_(—)003098), and 25% identityfrom amino acids 99-511 with a protein from Nostoc sp. PCC 7120 that isan ABC transporter ATP-binding protein (Accession Nos. NP_(—)490403.1;NC_(—)003276).

A Gapped BlastP sequence alignment showed that SEQ ID NO:132 (529 aminoacids) has about 25% identity from amino acids 10-526 with a proteinfrom Lactococcus lactis subsp. lactis that is an ABC transporter ATPbinding and permease protein (Accession Nos. NP_(—)267678.1;NC_(—)002662), about 26% identity from amino acids 112-525 with aprotein from Streptococcus pneumoniae that is an ABC transporterATP-binding/membrane spanning permease (Accession Nos. NP_(—)357731.1;NC_(—)003098), about 26% identity from amino acids 112-525 with aprotein from Streptococcus pneumoniae that is an ABC transporter,ATP-binding protein (Accession Nos. NP_(—)344680.1; NC_(—)003028), about24% identity from amino acids 107-518 with a protein from Brevibacillusbrevis that is homologous to an ABC-transporter (TycD) (Accession No.pir∥T31077), and 24% identity from amino acids 83-521 with a proteinfrom Streptococcus pneumoniae that is a drug efflux ABC transporter,ATP-binding/permease (Accession Nos. NP_(—)345800.1; NC_(—)003028).

A Gapped BlastP sequence alignment showed that SEQ ID NO:134 (600 aminoacids) has about 23% identity from amino acids 2-600 with a protein fromListeria innocua that is homologous to an ABC transporter (permease)(Accession Nos. NP_(—)471553.1; NC_(—)003212), about 23% identity fromamino acids 1-598 with a protein from Listeria monocytogenes that ishomologous to an ABC transporter (permease) (Accession Nos.NP_(—)465271.1; NC_(—)003210), about 22% identity from amino acids 1-599with a protein from Clostridium perfringens that is homologous to an ABCtransporter (Accession Nos. NP_(—)561767.1; NC_(—)003366), about 22%identity from amino acids 1-564 with a protein from Clostridiumperfringens that is homologous to an ABC-transporter (Accession Nos.NP_(—)561039.1; NC_(—)003366), and 22% identity from amino acids 4-593with a protein from Clostridium acetobutylicum that is homologous to apermease (Accession Nos. NP_(—)346868.1; NC_(—)003030).

A Gapped BlastP sequence alignment showed that SEQ ID NO:136 (249 aminoacids) has about 58% identity from amino acids 1-242 with a protein fromClostridium perfringens that is homologous to an ABC transporter(Accession Nos. NP_(—)561766.1; NC_(—)003366), about 55% identity fromamino acids 3-242 with a protein from Clostridium perfringens that ishomologous to an ABC transporter (Accession Nos. NP_(—)561038.1;NC_(—)003366), about 51% identity from amino acids 1-242 with a proteinfrom Listeria monocytogenes that is homologous to an ABC transporter(ATP-binding protein) (Accession Nos. NP_(—)465638.1; NC_(—)003210),about 50% identity from amino acids 1-242 with a protein from Listeriainnocua that is homologous to an ABC-transporter (ATP-binding protein)(Accession Nos. NP_(—)471552.1; NC_(—)003212), and 54% identity fromamino acids 3-242 with a protein from Clostridium acetobutylicum that isan ABC transporter, ATP-binding protein (Accession Nos. NP_(—)346867.1;NC_(—)003030).

A Gapped BlastP sequence alignment showed that SEQ ID NO:138 (423 aminoacids) has about 21% identity from amino acids 2-391 with a hypotheticalprotein from Streptococcus pyogenes (Accession Nos. NP_(—)270004.1;NC_(—)002737), about 21% identity from amino acids 2-383 with ahypothetical protein from Streptococcus pyogenes (Accession Nos.NP_(—)608080.1; NC_(—)003485), about 26% identity from amino acids 9-166with a protein from Bacillus subtilis that is a yvbJ protein (AccessionNos. NP_(—)391268.1; NC_(—)000964), about 25% identity from amino acids92-281 with a protein from caprine arthritis-encephalitis virus that isan env polyprotein precursor (Accession No. pir∥VCLJC6), and 24%identity from amino acids 92-281 with a protein from Caprinearthritis-encephalitis virus that is an envelope glycoprotein (AccessionNos. gb|AAD14661.1; AF105181).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:140(438 amino acids) has about 27% identity from amino acids 86-216 with aprotein from Brochothrix campestris that is a transport accessoryprotein (Accession Nos. gb|AAC95141.1; AF075600), about 26% identityfrom amino acids 107-219 with a protein from Streptococcus pneumoniaethat is a bacterocin transport accessory protein (Accession Nos.NP_(—)345950.1; NC_(—)003028), about 26% identity from amino acids107-219 with a protein from Streptococcus pneumoniae that is a Bta(Accession Nos. gb|AAD56628.1; AF165218), 23% identity from amino acids88-201 with a hypothetical protein from Bacillus anthracis (AccessionNos. NP_(—)052783.1; NC_(—)001496), and 32% identity from amino acids144-214 with a protein from Neisseria meningitidis that is a thioredoxin(Accession Nos. NP_(—)274384.1; NC_(—)003112).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:142(196 amino acids) has about 56% identity from amino acids 1-196 with aprotein from Lactobacillus gasseri (Accession Nos. dbj|BAA82351.1;AB029612), about 49% identity from amino acids 10-196 with ahypothetical protein from Lactobacillus sp. (Accession No.sp|P29470|YLA1_LACAC), about 28% identity from amino acids 41-196 with aprotein from Lactobacillus casei that is an ABC-transporter accessoryfactor (Accession Nos. NP_(—)542220.1; NC_(—)003320), 35% identity fromamino acids 90-196 with a protein from Lactobacillus plantarum that isan accessory factor for ABC-transporter (PlnH) (Accession Nos.emb|CAA64190.1; X94434), and 30% identity from amino acids 41-196 with aprotein from Lactobacillus sake that is homologous to an ABC exporteraccessory factor (SapE) (Accession No. pir∥A56973).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:144(720 amino acids) has about 62% identity from amino acids 9-720 with aprotein from Lactobacillus plantarum that is an ABC-transporter (PlnG)(Accession Nos. emb|CAA64189.1; X94434), about 62% identity from aminoacids 6-720 with a protein from Lactobacillus sakei that is homologousto a translocation protein (sppT), ATP-dependent (Accession No.pir∥S57913), about 62% identity from amino acids 2-720 with a proteinfrom Lactobacillus sakei that is an ATP-dependent transport protein(SapT) (Accession No. pir∥I56273), 62% identity from amino acids 9-720with a protein from Lactobacillus casei that is an ABC transporter(Accession Nos. NP_(—)542219.1; NC_(—)003320), and 57% identity fromamino acids 25-718 with a protein from Lactobacillus acidophilus that isan ABC transporter (Accession Nos. NP_(—)604412.1; NC_(—)003458).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:146(234 amino acids) has about 52% identity from amino acids 13-228 with aprotein from Staphylococcus aureus subsp. aureus that is homologous toan ABC transporter ATP-binding protein (Accession Nos. NP_(—)370833.1;NC_(—)002758), about 50% identity from amino acids 11-234 with a proteinfrom Streptococcus pyogenes that is homologous to an ABC transporter(ATP-binding protein) (Accession Nos. NP_(—)606994.1; NC_(—)003485),about 50% identity from amino acids 11-234 with a protein fromStreptococcus pyogenes that is homologous to an ABC transporter(ATP-binding protein) (Accession Nos. NP_(—)268993.1; NC_(—)002737), 50%identity from amino acids 13-232 with a protein from Lactococcus lactissubsp. lactis that is an ABC transporter ATP-binding protein (AccessionNos. NP_(—)266815.1; NC_(—)002662), and 53% identity from amino acids11-233 with a protein from Lactococcus lactis subsp. lactis that is anABC transporter ATP-binding protein (Accession Nos. NP_(—)268413.1;NC_(—)002662).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:148(353 amino acids) has about 40% identity from amino acids 1-352 with ahypothetical protein from Lactococcus lactis subsp. lactis (AccessionNos. NP_(—)268412.1; NC_(—)002662), about 38% identity from amino acids1-352 with a conserved hypothetical protein from Staphylococcus aureussubsp. aureus (Accession Nos. NP_(—)370832.1; NC_(—)002758), about 33%identity from amino acids 1-352 with a conserved hypothetical proteinfrom Streptococcus pyogenes (Accession Nos. NP_(—)268992.1;NC_(—)002737), 33% identity from amino acids 1-352 with a conservedhypothetical protein from Streptococcus pyogenes (Accession Nos.NP_(—)606993.1; NC_(—)003485), and 34% identity from amino acids 1-352with a protein from Lactococcus lactis subsp. lactis that is an ABCtransporter permease protein (Accession Nos. NP_(—)266816.1;NC_(—)002662).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:150(188 amino acids) has about 47% identity from amino acids 14-85 with aprotein from Lactococcus lactis subsp. lactis that is a transcriptionalregulator (Accession Nos. NP_(—)266817.1; NC_(—)002662), about 28%identity from amino acids 21-90 with a protein from Aquifex aeolicusthat is a transcriptional regulator in the TetR/AcrR family (AccessionNos. NP_(—)213195.1; NC_(—)000918), about 30% identity from amino acids14-75 with a protein from Clostridium acetobutylicum that is atranscriptional regulator in the AcrR family (Accession Nos.NP_(—)348163.1; NC_(—)003030), 29% identity from amino acids 25-109 witha protein from Streptomyces coelicolor that is homologous to atranscriptional regulator (Accession Nos. emb|CAB93030.1; AL357432), and41% identity from amino acids 27-88 with a protein from Clostridiumacetobutylicum that is a transcriptional regulator in the TetR/AcrRfamily (Accession Nos. NP_(—)347457.1; NC_(—)003030).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:152(236 amino acids) has about 65% identity from amino acids 3-236 with aprotein from Streptococcus pneumoniae that is an ABC transporterATP-binding protein (Accession Nos. NP_(—)359090.1; NC_(—)003098), about66% identity from amino acids 4-236 with a protein from Streptococcuspneumoniae that is an ABC transporter, ATP-binding protein (AccessionNos. NP_(—)346092.1; NC_(—)003028), about 65% identity from amino acids4-236 with a protein from Streptococcus pyogenes that is homologous toan ABC transporter (ATP-binding protein) (Accession Nos. NP_(—)607321.1;NC_(—)003485), 65% identity from amino acids 4-236 with a protein fromStreptococcus pyogenes that is homologous to an ABC transporter(ATP-binding protein) (Accession Nos. NP_(—)269390.1; NC_(—)002737), and62% identity from amino acids 4-236 with a protein from Listeriamonocytogenes that is homologous to a ABC transporter, ATP-bindingprotein (Accession Nos. NP_(—)464748.1; NC_(—)003210).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:154(846 amino acids) has about 41% identity from amino acids 6-846 with aprotein from Lactococcus lactis subsp. lactis that is an ABC transporterpermease protein (Accession Nos. NP_(—)267260.1; NC_(—)002662), about34% identity from amino acids 2-846 with a hypothetical protein fromStreptococcus pneumoniae (Accession Nos. NP_(—)359089.1; NC_(—)003098),about 34% identity from amino acids 2-846 with a hypothetical proteinfrom Streptococcus pneumoniae (Accession Nos. NP_(—)346091.1;NC_(—)003028), 33% identity from amino acids 4-846 with a hypotheticalprotein from Streptococcus pyogenes (Accession Nos. NP_(—)269389.1;NC_(—)002737), and 33% identity from amino acids 4-846 with ahypothetical protein from Streptococcus pyogenes (Accession Nos.NP_(—)607320.1; NC_(—)003485).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:156(78 amino acids) has about 30% identity from amino acids 12-70 with aprotein from Arabidopsis thaliana (Accession Nos. gb|AAF19707.1;AC008047), about 30% identity from amino acids 12-70 with a protein fromArabidopsis thaliana that is homologous to an ATP dependent coppertransporter (Accession Nos. NP_(—)176533.1; NM_(—)105023), about 32%identity from amino acids 1-65 with a hypothetical protein fromPyrococcus furiosus (Accession Nos. NP_(—)579673.1; NC_(—)003413), and37% identity from amino acids 21-55 with a protein from Hepatitis TTvirus (Accession Nos. gb|AAK11712.1; AF345529).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:158(379 amino acids) has about 36% identity from amino acids 32-368 with aconserved hypothetical protein from Listeria innocua (Accession Nos.NP_(—)470340.1; NC_(—)003212), about 37% identity from amino acids32-353 with a conserved hypothetical protein from Listeria monocytogenes(Accession Nos. NP_(—)464529.1; NC_(—)003210), about 36% identity fromamino acids 87-370 with a protein from Lactococcus lactis (AccessionNos. emb|CAA68042.1; X99710), 31% identity from amino acids 28-372 witha hypothetical protein from Lactococcus lactis subsp. lactis (AccessionNos. NP_(—)267885.1; NC_(—)002662), and 30% identity from amino acids32-348 with a protein from Actinosynnema pretiosum subsp. auranticum(Accession Nos. gb|AAC14002.1; U33059).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:160(779 amino acids) has about 61% identity from amino acids 1-308 with aprotein from Streptococcus mutans that is an ABC transporter ATP bindingsubunit (Accession Nos. gb|AAD09218.1; U73183), about 37% identity fromamino acids 1-362 with a protein from Lactococcus lactis subsp. lactisthat is an ABC transporter ATP-binding and permease protein (AccessionNos. NP_(—)266870.1; NC_(—)002662), about 39% identity from amino acids1-295 with a protein from Listeria monocytogenes that is homologous toan ABC transporter, ATP-binding protein (Accession Nos. NP_(—)464271.1;NC_(—)003210), 47% identity from amino acids 1-221 with a protein fromArchaeoglobus fulgidus that is an ABC transporter, ATP-binding protein(Accession Nos. NP_(—)070298.1; NC_(—)000917), and 49% identity fromamino acids 1-218 with a protein from Archaeoglobus fulgidus that is anABC transporter, ATP-binding protein (Accession Nos. NP_(—)069851.1;NC_(—)000917).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:162(38 amino acids) has about 66% identity from amino acids 1-27 with aprotein from Clostridium acetobutylicum that is a mannose-specificphosphotransferase system component (Accession Nos. NP_(—)149230.1;NC_(—)001988), about 72% identity from amino acids 3-27 with a proteinfrom Listeria monocytogenes that is homologous to a PTS systemmannose-specific factor IIAB (Accession Nos. NP_(—)463629.1;NC_(—)003210), about 72% identity from amino acids 3-27 with a proteinfrom Listeria innocua that is homologous to a PTS systemmannose-specific factor IIAB (Accession Nos. NP_(—)469488.1;NC_(—)003212), 66% identity from amino acids 1-27 with a protein fromClostridium perfringens that is a PTS system protein (Accession Nos.NP_(—)561737.1; NC_(—)003366), and 65% identity from amino acids 2-27with a protein from Streptococcus pyogenes that is a mannose-specificphosphotransferase system component IIAB (Accession Nos. NP_(—)269761.1;NC_(—)002737).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:164(105 amino acids) has about 60% identity from amino acids 1-103 with aprotein from Listeria monocytogenes that is homologous to a PTS systemmannose-specific factor IIAB (Accession Nos. NP_(—)463629.1;NC_(—)003210), about 59% identity from amino acids 1-103 with a proteinfrom Listeria innocua that is homologous to a PTS systemmannose-specific factor IIAB (Accession Nos. NP_(—)469488.1;NC_(—)003212), about 57% identity from amino acids 1-104 with a proteinfrom Clostridium perfringens that is a PTS system protein (AccessionNos. NP_(—)561737.1; NC_(—)003366), 53% identity from amino acids 1-104with a protein from Clostridium acetobutylicum that is amannose-specific phosphotransferase system component IIAB (AccessionNos. NP_(—)149230.1; NC_(—)001988), and 54% identity from amino acids1-96 with a protein from Streptococcus pyogenes that is amannose-specific phosphotransferase system component IIAB (AccessionNos. NP_(—)607831.1; NC_(—)003485).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:166(269 amino acids) has about 69% identity from amino acids 1-269 with aprotein from Listeria innocua that is homologous to a PTS systemmannose-specific, factor IIC (Accession Nos. NP_(—)469489.1;NC_(—)003212), about 69% identity from amino acids 1-269 with a proteinfrom Listeria monocytogenes that is homologous to a PTS systemmannose-specific, factor IIC (Accession Nos. NP_(—)463630.1;NC_(—)003210), about 67% identity from amino acids 1-269 with a proteinfrom Streptococcus pneumoniae that is a PTS system, mannose-specific IICcomponent (Accession Nos. NP_(—)344821.1; NC_(—)003028), 65% identityfrom amino acids 1-269 with a protein from Streptococcus pyogenes thatis homologous to a mannose-specific phosphotransferase system componentTIC (Accession Nos. NP_(—)269762.1; NC_(—)002737), and 64% identity fromamino acids 1-269 with a protein from Clostridium acetobutylicum that isa mannose/fructose-specific phosphotransferase system component TIC(Accession Nos. NP_(—)149231.1; NC_(—)001988).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:168(307 amino acids) has about 67% identity from amino acids 5-307 with aprotein from Listeria innocua that is homologous to a PTS systemmannose-specific factor IID (Accession Nos. NP_(—)469490.1;NC_(—)003212), about 67% identity from amino acids 5-307 with a proteinfrom Listeria monocytogenes that is homologous to a PTS systemmannose-specific factor IID (Accession Nos. NP_(—)463631.1;NC_(—)003210), about 64% identity from amino acids 6-303 with a proteinfrom Clostridium acetobutylicum that is a mannose-specificphosphotransferase system component IID (Accession Nos. NP_(—)149232.1;NC_(—)001988), 64% identity from amino acids 4-300 with a protein fromLactococcus lactis subsp. lactis that is a mannose-specific PTS systemcomponent IID (EC 2.7.1.69) (Accession Nos. NP_(—)267864.1;NC_(—)002662), and 64% identity from amino acids 5-307 with a proteinfrom Streptococcus pneumoniae that is a PTS system, mannose-specific IIDcomponent (Accession Nos. NP_(—)344820.1; NC_(—)003028).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:170(111 amino acids) has about 51% identity from amino acids 4-105 with aprotein from Streptococcus pyogenes that is homologous to a PTS systemenzyme II protein (Accession Nos. NP_(—)269441.1; NC_(—)002737), about54% identity from amino acids 4-110 with a protein from Listeriamonocytogenes that is homologous to a cellobiose phosphotransferaseenzyme IIB component (Accession Nos. NP_(—)466205.1; NC_(—)003210),about 54% identity from amino acids 4-110 with a protein from Listeriainnocua that is homologous to a cellobiose phosphotransferase enzyme IIBcomponent (Accession Nos. NP_(—)472159.1; NC_(—)003212), 50% identityfrom amino acids 4-105 with a protein from Streptococcus pyogenes thatis homologous to a PTS system enzyme II (Accession Nos. NP_(—)607438.1;NC_(—)003485), and 50% identity from amino acids 1-109 with a proteinfrom Lactococcus lactis subsp. lactis that is a cellobiose-specific PTSsystem IIB component (EC 2.7.1.69) (Accession Nos, NP_(—)266569.1;NC_(—)002662).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:172(256 amino acids) has about 53% identity from amino acids 1-250 with aprotein from Streptococcus pneumoniae that is a phosphotransferasesystem sugar-specific EII component (Accession Nos. NP_(—)357876.1;NC_(—)003098), about 53% identity from amino acids 1-250 with a proteinfrom Streptococcus pneumoniae that is a PTS system IIC component(Accession Nos. NP_(—)344847.1; NC_(—)003028), about 43% identity fromamino acids 1-255 with a protein from Clostridium acetobutylicum that isa PTS cellobiose-specific component IIC (Accession Nos. NP_(—)347026.1;NC_(—)003030), 38% identity from amino acids 1-249 with a protein fromLactococcus lactis subsp. lactis that is a cellobiose-specific PTSsystem IIC component (EC 2.7.1.69) (Accession Nos. NP_(—)266572.1;NC_(—)002662), and 37% identity from amino acids 1-255 with a proteinfrom Listeria innocua that is homologous to a PTS system,cellobiose-specific IIC component (Accession Nos. NP_(—)470241.1;NC_(—)003212).

A Gapped BlastP (version) sequence alignment showed that SEQ ID NO:174(560 amino acids) has about 39% identity from amino acids 1-551 with aprotein from Bacillus halodurans that is a PTS system,beta-glucoside-specific enzyme II, ABC component (Accession Nos.NP_(—)241162.1; NC_(—)002570), about 39% identity from amino acids 1-551with a protein from Listeria monocytogenes that is homologous to aphosphotransferase system (PTS) beta-glucoside-specific enzyme IIABCcomponent (Accession Nos. NP_(—)464265.1; NC_(—)003210), about 38%identity from amino acids 1-554 with a protein from Bacillus subtilisthat is a phosphotransferase system (PTS) beta-glucoside-specific enzymeIIABC component (Accession Nos. NP_(—)391806.1; NC_(—)000964), 38%identity from amino acids 1-554 with a protein from Bacillus subtilisthat is a PTS system, beta-glucoside-specific IIABC component(EIIABC-BGL) (beta-glucoside-permease IIABC component) (Accession No.sp|P40739|PTBA_BACSU), and 37% identity from amino acids 1-554 with aprotein from Bacillus halodurans that is a PTS system,beta-glucoside-specific enzyme II, ABC component (Accession Nos.NP_(—)241461.1; NC_(—)002570).

The top blast result for even SEQ ID NOS:176-308 is shown in Table 2.

TABLE 2 Top Blast result for SEQ ID NOS: 176-308 SEQ Amino ID PercentAcid NO: ORF Identity Range Organism Description Accession No. 176 146383 3 to Lactobacillus lactose permease emb|CAD55501.1 639 helveticus 178639 90 1 to 88 Lactobacillus phosphocarrier ref|NP_964671.1 johnsoniiNCC 533 protein HPr 180 640 83 1 to Lactobacillus phosphoenolpyruvate-ref|NP_964672.1 576 johnsonii NCC 533 protein phosphotransferase (enzymeI) 182 431 77 1 to Lactobacillus pepR1 emb|CAB76946.1 333 delbrueckiisubsp. bulgaricus 184 676 71 1 to Lactobacillus HPr(Ser) ref|NP_964704.1314 johnsonii NCC 533 kinase/phosphatase 186 1778 79 1 to Lactobacillusfructose-1- ref|NP_965684.1 303 johnsonii NCC 533 phosphate kinase 1881779 54 1 to Lactobacillus ref|NP_965685.1 251 johnsonii NCC 533 1901433 77 1 to Lactobacillus glycerone kinase ref|NP_784000.1 331plantarum WCFS1 192 1434 64 3 to Lactobacillus dihydroxyacetoneref|NP_784001.1 194 plantarum WCFS1 kinase, phosphatase domain dak2 1941436 73 1 to Lactobacillus glycerol uptake ref|NP_784003.1 231 plantarumWCFS1 facilitator protein 196 1437 100 1 to Lactobacillus sucrosegb|AAO21868.1 480 acidophilus phosphorylase 198 1438 100 1 toLactobacillus alpha-galactosidase gb|AAO21867.1 732 acidophilus 200 145774 1 to Lactobacillus aldose 1-epimerase ref|NP_964716.1 327 johnsoniiNCC 533 202 1458 84 1 to Lactobacillus galactose-1-P- emb|CAA40526.1 486helveticus uridyl transferase 204 1459 89 1 to Lactobacillusgalactokinase emb|CAA40525.1 387 helveticus 206 1460 31 79 toLactobacillus cell surface protein ref|NP_784891.1 305 plantarum WCFS1precursor 208 1461 27 2 to Lactobacillus ref|NP_964254.1 201 johnsoniiNCC 533 210 1462 74 1 to Lactobacillus beta-galactosidaseref|NP_964713.1 665 johnsonii NCC 533 212 1467 99 1 to Lactobacillusbeta-galactosidase dbj|BAA20536.1 628 acidophilus 214 1468 100 1 toLactobacillus BGAM_LACAC sp|O07685 316 acidophilus beta-galactosidasesmall subunit (LACTASE) 216 1469 95 1 to Lactobacillus UDP-galactose 4-emb|CAD55502.1 330 helveticus epimerase 218 1719 80 1 to LactobacillusUTP--glucose-1- ref|NP_965397.1 294 johnsonii NCC 533 phosphateuridylyltransferase 220 874 87 6 to Lactobacillus JE0395 phospho-pir||JE0395 481 gasseri beta-galactosidase I- Lactobacillus gasseri 222910 66 3 to Lactobacillus COG0039: ref|ZP_00046547.1 308 gasseriMalate/lactate dehydrogenases 224 1007 55 13 to Lactobacillus COG2240:ref|ZP_00046499.1 279 gasseri Pyridoxal/pyridoxine/ pyridoxamine kinase226 1812 71 3 to Lactobacillus alpha-glucosidase ref|NP_965686.1 766johnsonii NCC 533 228 1632 69 1 to Lactobacillus succinate-ref|NP_965584.1 457 johnsonii NCC 533 semialdehyde dehydrogenase 2301401 89 1 to Lactobacillus COG0446: ref|ZP_00046159.1 454 gasseriUncharacterized NAD(FAD)- dependent dehydrogenases 232 1974 72 1 toacetolactate COG0028: ref|ZP_00047198.1 601 synthase, pyruvate Thiaminedehydrogenase pyrophosphate- (cytochrome), requiring enzymes glyoxylatecarboligase, phosphonopyruvate decarboxylase 234 1102 56 1 toLactobacillus transmembrane emb|CAA05490.1 269 helveticus protein 2361783 68 1 to Lactobacillus ABC transporter ref|NP_965688.1 298 johnsoniiNCC 533 ATPase component 238 1879 72 9 to Lactobacillus COG0351:ref|ZP_00046866.1 268 gasseri Hydroxymethylpyrimidine/phosphomethylpyrimidine kinase 240 680 56 8 to Streptococcusref|NP_735321.1 633 agalactiae NEM316 242 55 96 8 to LactobacillusCOG1052: Lactate ref|ZP_00046778.2 349 gasseri dehydrogenase and relateddehydrogenases 244 185 97 1 to Lactobacillus COG0588: ref|ZP_00047243.1230 gasseri Phosphoglycerate mutase 1 246 271 91 1 to Lactobacilluslactate emb|CAB03618.1 323 helveticus dehydrogenase 248 698 92 1 toLactobacillus glyceraldehyde 3- ref|NP_964727.1 338 johnsonii NCC 533phosphate dehydrogenase 250 699 93 1 to Lactobacillus phosphoglycerateref|NP_964728.1 403 johnsonii NCC 533 kinase 252 752 83 3 toLactobacillus COG0166: ref|ZP_00046229.1 445 gasseri Glucose-6-phosphate isomerase 254 889 93 1 to Lactobacillus COG0148: Enolaseref|ZP_00046557.1 428 gasseri 256 956 78 1 to Lactobacillus 6-ref|NP_964935.1 319 johnsonii NCC 533 phosphofructokinase 258 957 88 1to Lactobacillus COG0469: ref|ZP_00046514.1 589 gasseri Pyruvate kinase260 1599 81 1 to Lactobacillus fructose- ref|NP_964539.1 303 johnsoniiNCC 533 bisphosphate aldolase 262 1641 71 1 to Lactobacillus COG1653:ABC- ref|ZP_00046816.2 433 gasseri type sugar transport system,periplasmic component 264 452 69 1 to Lactobacillus phosphoenolpyruvate-ref|NP_965752.1 335 johnsonii NCC 533 dependent sugar phosphotransferasesystem 266 1479 71 1 to Lactobacillus ref|NP_965117.1 278 johnsonii NCC533 268 725 62 1 to Lactobacillus COG1263: ref|ZP_00046302.1 655 gasseriPhosphotransferase system IIC components, 270 1369 81 1 to Lactobacillusphosphoenolpyruvate- ref|NP_964585.1 411 johnsonii NCC 533 dependentsugar phosphotransferase system EIIC, 272 227 52 1 to Enterococcus PTSsystem, IIC ref|NP_814084.1 436 faecalis V583 component 274 502 100 1 toLactobacillus substrate-binding gb|AAO21856.1 431 acidophilus proteinMsmE 276 507 100 1 to Lactobacillus sucrose gb|AAO21861.1 480acidophilus phosphorylase 278 1483 59 1 to Streptococcus ref|NP_734585.1492 agalactiae NEM316 280 1484 75 1 to Lactobacillus high affinityribose ref|NP_965069.1 131 johnsonii NCC 533 transport protein rbsD 282552 76 1 to Lactobacillus major facilitator ref|NP_964553.1 487johnsonii NCC 533 superfamily permease 284 567 79 3 to LactobacillusCOG0477: ref|ZP_00045998.1 400 gasseri Permeases of the majorfacilitator superfamily 286 1471 74 79 to Lactobacillus ref|NP_965113.1405 johnsonii NCC 533 288 1853 80 4 to Lactobacillus COG0477:ref|ZP_00046596.1 163 gasseri Permeases of the major facilitatorsuperfamily 290 1012 77 9 to Lactobacillus phosphoenolpyruvate-ref|NP_964612.1 643 johnsonii NCC 533 dependent sugar phosphotransferasesystem 292 1014 77 1 to Lactobacillus COG0366: ref|ZP_00045981.1 552gasseri Glycosidases 294 1440 100 1 to Lactobacillus transmembranegb|AAO21865.1 277 acidophilus permease MsmG2 296 1442 100 1 toLactobacillus substrate-binding gb|AAO21863.1 418 acidophilus proteinMsmE2 298 1132 62 1 to Lactobacillus COG1132: ABC- ref|ZP_00045932.1 525gasseri type multidrug transport system, ATPase and permease 300 1358 371 to Lactobacillus COG1132: ABC- ref|ZP_00045932.1 525 gasseri typemultidrug transport system, ATPase and permease 302 1838 71 1 toLactobacillus ABC transporter ref|NP_965714.1 224 johnsonii NCC 533ATPase component 304 1840 50 1 to Lactobacillus ref|NP_965716.1 172johnsonii NCC 533 306 1913 72 1 to Lactobacillus COG1136: ABC-ref|ZP_00045892.1 233 gasseri type antimicrobial peptide transportsystem, ATPase 308 1938 59 19 to Lactobacillus ref|NP_965786.1 364johnsonii NCC 533

Example 2 PFAM Results for Amino Acid Sequences

Table 3 shows the top PFAM results for the amino acid sequences of theinvention.

TABLE 3 PFAM Results for Amino Acid Sequences Amino Acid SEQ ID RangePFAM NO: ORF Domain Start, Stop Family Accession No. E-value 3 877PTS_IIA 16, 111 PTS system, Lactose/Cellobiose specific IIA PF022558.20E−40 subunit 5 609 PTS_EIIA_1 30, 134 phosphoenolpyruvate-dependentsugar PF00358 6.00E−55 phosphotransferase system, EIIA 1 7 1479 PRD 76,171; PRD domain PF00874 9.90E−52 181, 282 7 1479 CAT_RBD 6, 67 CAT RNAbinding domain PF03123 1.10E−16 9 1574 Glyco_hydro_1 4, 471 Glycosylhydrolase family 1 PF00232 2.90E−133 11 1707 PTS_EIIA_1 491, 595phosphoenolpyruvate-dependent sugar PF00358 6.10E−53 phosphotransferasesystem, EIIA 1 11 1707 PTS_EIIC 105, 387 Phosphotransferase system, EIICPF02378 3.10E−33 11 1707 PTS_EIIB 7, 41 phosphotransferase system, EIIBPF00367 8.50E−19 13 725 PTS_EIIA_1 528, 632phosphoenolpyruvate-dependent sugar PF00358 4.10E−60 phosphotransferasesystem, EIIA 1 13 725 PTS_EIIC 122, 419 Phosphotransferase system, EIICPF02378 3.80E−35 13 725 PTS_EIIB 21, 55 phosphotransferase system, EIIBPF00367 8.90E−17 15 491 PTS_EIIC 35, 368 Phosphotransferase system, EIICPF02378 6.90E−80 19 1684 EIIA-man 1, 115 PTS system fructose IIAcomponent PF03610 1.20E−13 27 884 PTS_EIIC 34, 392 Phosphotransferasesystem, EIIC PF02378 7.70E−86 29 618 PTS_EIIC 29, 360 Phosphotransferasesystem, EIIC PF02378 8.70E−40 31 606 PTS_EIIC 9, 351 Phosphotransferasesystem, EIIC PF02378 3.10E−48 31 606 PTS_EIIB 457, 491;phosphotransferase system, EIIB PF00367 1.40E−22 551, 585 33 1705PTS_EIIA_1 531, 636 phosphoenolpyruvate-dependent sugar PF00358 3.90E−48phosphotransferase system, EIIA 1 33 1705 PTS_EIIC 131, 412Phosphotransferase system, EIIC PF02378 2.80E−38 33 1705 PTS_EIIB 10, 44phosphotransferase system, EIIB PF00367 2.00E−13 35 1777 PTS_IIB_fruc183, 285 PTS system, Fructose specific IIB subunit PF02379 2.40E−45 351777 PTS_EIIC 313, 597 Phosphotransferase system, EIIC PF02378 5.20E−3435 1777 PTS_EIIA_2 5, 149 Phosphoenolpyruvate-dependent sugar PF003592.60E−26 phosphotransferase system, EIIA 2 37 500 Peripla_BP_1 68, 331Periplasmic binding proteins and sugar PF00532 2.60E−10 binding domainof the LacI family 37 500 LacI 11, 36 Bacterial regulatory proteins,lacI family PF00356 6.40E−10 39 502 SBP_bacterial_1 28, 403 Bacterialextracellular solute-binding protein PF01547 1.50E−51 41 503BPD_transp_1 66, 287 Binding-protein-dependent transport system PF005282.30E−19 inner membrane component 43 504 BPD_transp_1 80, 279Binding-protein-dependent transport system PF00528 1.00E−19 innermembrane component 45 505 Glyco_hydro_32 24, 409 Glycosyl hydrolasesfamily 32 PF00251 5.50E−72 47 506 ABC_tran 31, 212 ABC transporterPF00005 2.70E−58 53 1482 BPD_transp_2 5, 274 Branched-chain amino acidtransport system/ PF02653 6.40E−73 permease component 55 1483 ABC_tran32, 219; ABC transporter PF00005 8.70E−88 280, 472 59 1485 PfkB 4, 297pfkB family carbohydrate kinase PF00294 4.70E−73 61 1864 BPD_transp_170, 280 Binding-protein-dependent transport system PF00528 5.80E−13inner membrane component 63 1865 BPD_transp_1 213, 451Binding-protein-dependent transport system PF00528 2.90E−13 innermembrane component 65 1866 SBP_bac_1 7, 322 Bacterial extracellularsolute-binding protein PF01547 1.40E−22 67 1867 ABC_tran 31, 212 ABCtransporter PF00005 8.00E−58 69 1944 ABC_tran 34, 220; ABC transporterPF00005 8.20E−64 287, 481 71 1945 BPD_transp_2 53, 338 Branched-chainamino acid transport system/ PF02653 1.40E−43 permease component 73 1946BPD_transp_2 10, 297 Branched-chain amino acid transport system/ PF026539.40E−44 permease component 79 566 Sugar_tr 25, 93 Sugar (and other)transporter PF00083 1.10E−10 89 1616 Polysacc_synt 16, 329Polysaccharide biosynthesis protein PF01943 1.70E−08 97 399 Peripla_BP_160, 325 Periplasmic binding proteins and sugar PF00532 1.60E−18 bindingdomain of the LacI family 97 399 LacI 3, 28 Bacterial regulatoryproteins, lacI family PF00356 8.50E−11 99 400 Glyco_hydro_32 37, 449Glycosyl hydrolases family 32 PF00251 5.40E−158 101 401 PTS_EIIA_1 517,621 phosphoenolpyruvate-dependent sugar PF00358 2.00E−70phosphotransferase system, EIIA 1 101 401 PTS_EIIC 111, 403Phosphotransferase system, EIIC PF02378 4.60E−68 101 401 PTS_EIIB 7, 40phosphotransferase system, EIIB PF00367 5.50E−14 103 1012 PTS_EIIA_1 49,153 phosphoenolpyruvate-dependent sugar PF00358 4.00E−45phosphotransferase system, EIIA 1 103 1012 PTS_EIIC 301, 587Phosphotransferase system, EIIC PF02378 1.20E−43 103 1012 PTS_EIIB 197,231 phosphotransferase system, EIIB PF00367 2.40E−16 105 1013 GntR 5, 68Bacterial regulatory proteins, gntR family PF00392 2.50E−15 107 1014Alpha-amylase 28, 429 Alpha amylase, catalytic domain PF00128 1.50E−110109 1439 ABC_tran 31, 212 ABC transporter PF00005 2.20E−58 109 1439 TOBE301, 359 TOBE domain PF03459 6.80E−09 111 1440 BPD_transp_1 162, 235Binding-protein-dependent transport system PF00528 2.90E−27 innermembrane component 113 1441 BPD_transp_1 66, 290Binding-protein-dependent transport system PF00528 1.60E−29 innermembrane component 115 1442 SBP_bacterial_1 48, 411 Bacterialextracellular solute-binding protein PF01547 1.20E−61 117 1443AraC_binding 16, 159 AraC-like ligand binding domain PF02311 6.80E−30117 1443 HTH_AraC 229, 273 Bacterial regulatory helix-turn-helix PF001658.40E−20 proteins, AraC family 121 74 ABC_tran 346, 527 ABC transporterPF00005 2.80E−36 123 75 ABC_tran 346, 527 ABC transporter PF000059.20E−35 125 1131 ABC_tran 346, 527 ABC transporter PF00005 4.50E−35 1251131 ABC_membrane 14, 280 ABC transporter transmembrane region PF006641.00E−08 127 1132 ABC_tran 347, 528 ABC transporter PF00005 4.80E−36 1291357 ABC_tran 346, 527 ABC transporter PF00005 3.50E−33 131 1358ABC_tran 348, 529 ABC transporter PF00005 1.30E−35 133 1679 FtsX 85, 182Predicted permease PF02687 1.40E−08 135 1680 ABC_tran 35, 221 ABCtransporter PF00005 1.90E−60 143 1796 ABC_membrane 164, 440 ABCtransporter transmembrane region PF00664 5.10E−68 143 1796 Peptidase_C3910, 145 Peptidase C39 family PF03412 3.30E−64 143 1796 ABC_tran 512, 696ABC transporter PF00005 1.60E−46 145 1838 ABC_tran 43, 228 ABCtransporter PF00005 2.10E−56 147 1839 FtsX 192, 347 Predicted permeasePF02687 4.80E−16 151 1913 ABC_tran 36, 217 ABC transporter PF000051.50E−57 153 1914 FtsX 246, 441; Predicted permease PF02687 7.60E−48668, 838 159 1939 ABC_tran 31, 216 ABC transporter PF00005 7.60E−57 1591939 FtsX 594, 772 Predicted permease PF02687 3.00E−35 165 455 EII-Sor1, 238 PTS system sorbose-specific iic component PF03609 8.00E−124 167456 EIID-AGA 7, 307 PTS system mannose/fructose/sorbose PF036134.80E−184 family IID component 169 876 PTS_IIB 5, 107 PTS system,Lactose/Cellobiose specific IIB PF02302 1.40E−31 subunit 173 1575PTS_EIIA_1 425, 529 phosphoenolpyruvate-dependent sugar PF00358 6.70E−63phosphotransferase system, EIIA 1 173 1575 PTS_EIIC 42, 322Phosphotransferase system, EIIC PF02378 1.40E−39 175 1463 PTS_EIIA_1516, 608 phosphoenolpyruvate-dependent sugar PF00358 2.10E−28phosphotransferase system, EIIA 1 177 639 PTS-HPr 1, 84 PTS HPrcomponent phosphorylation site PF00381 7.10E−52 179 640 PEP-utilizers_C252, 544 PEP-utilising enzyme, TIM barrel domain PF02896 8.30E−182 179640 PEP-utilisers_N 5, 129 PEP-utilising enzyme, N-terminal PF055243.50E−57 179 640 PEP-utilizers 146, 227 PEP-utilising enzyme, mobiledomain PF00391 4.60E−37 181 431 LacI 6, 31 Bacterial regulatoryproteins, lacI family PF00356 1.90E−11 183 676 Hpr_kinase_C 133, 313 HPrSerine kinase C-terminus PF07475 3.50E−86 183 676 Hpr_kinase_N 3, 132HPr Serine kinase N terminus PF02603 7.90E−26 185 1778 PfkB 7, 292 pfkBfamily carbohydrate kinase PF00294 1.50E−37 187 1779 DeoR 6, 231Bacterial regulatory proteins, deoR family PF00455 1.30E−64 189 1433Dak1 16, 331 Dak1 domain PF02733 2.30E−104 191 1434 Dak2 32, 189 DAK2domain PF02734 4.10E−71 193 1436 MIP 1, 231 Major intrinsic proteinPF00230 2.00E−39 195 1437 Alpha-amylase 10, 423 Alpha amylase, catalyticdomain PF00128 3.60E−07 197 1438 Melibiase 293, 690 Melibiase PF020659.00E−252 199 1457 Aldose_epim 18, 326 Aldose 1-epimerase PF012637.30E−63 201 1458 GalP_UDP_tr_C 222, 430 Galactose-1-phosphate uridyltransferase, C- PF02744 9.30E−106 terminal domain 201 1458GalP_UDP_transf 15, 220 Galactose-1-phosphate uridyl transferase,PF01087 2.30E−95 N-terminal domain 203 1459 GHMP_kinases 112, 351 GHMPkinases putative ATP-binding protein PF00288 2.00E−50 209 1462Glyco_hydro_42 192, 605 Beta-galactosidase PF02449 1.90E−150 211 1467Glyco_hydro_2_C 333, 628 Glycosyl hydrolases family 2, TIM barrelPF02836 2.60E−146 domain 211 1467 Glyco_hydro_2_N 39, 227 Glycosylhydrolases family 2, sugar binding PF02837 2.00E−86 domain 211 1467Glyco_hydro_2 229, 331 Glycosyl hydrolases family 2, PF00703 2.90E−21immunoglobulin-like beta-sandwich domain 213 1468 Bgal_small_N 4, 197Beta galactosidase small chain, N terminal PF02929 3.30E−94 domain 2131468 Bgal_small_C 206, 315 Beta galactosidase small chain, C terminalPF02930 8.40E−61 domain 215 1469 Epimerase 3, 324 NAD dependentepimerase/dehydratase PF01370 2.00E−142 family 215 1469 3Beta_HSD 2, 3243-beta hydroxysteroid PF01073 1.00E−07 dehydrogenase/isomerase family217 1719 NTP_transferase 5, 272 Nucleotidyl transferase PF00483 8.30E−28219 874 Glyco_hydro_1 2, 479 Glycosyl hydrolase family 1 PF002321.20E−136 221 910 Ldh_1_N 3, 142 lactate/malate dehydrogenase, NADbinding PF00056 1.60E−59 domain 221 910 Ldh_1_C 144, 308 lactate/malatedehydrogenase, alpha/beta PF02866 1.90E−32 C-terminal domain 223 1007PfkB 130, 187; pfkB family carbohydrate kinase PF00294 3.10E−07 217, 247225 1812 Glyco_hydro_31 105, 757 Glycosyl hydrolases family 31 PF010552.10E−120 227 1632 Aldedh 3, 456 Aldehyde dehydrogenase family PF001711.40E−98 229 1401 Pyr_redox 5, 294 Pyridine nucleotide-disulphidePF00070 2.10E−65 oxidoreductase 231 1974 TPP_enzyme_N 4, 174 Thiaminepyrophosphate enzyme, N- PF02776 4.00E−34 terminal TPP binding domain231 1974 TPP_enzyme_M 193, 340 Thiamine pyrophosphate enzyme, centralPF00205 1.40E−32 domain 233 1102 Sugar_transport 16, 280 Sugar transportprotein PF06800 1.60E−114 235 1783 ABC_tran 27, 204 ABC transporterPF00005 2.20E−44 237 1879 PfkB 129, 178; pfkB family carbohydrate kinasePF00294 5.50E−07 212, 240 239 680 Isoamylase_N 25, 102 IsoamylaseN-terminal domain PF02922 8.80E−19 239 680 Alpha-amylase 146, 495 Alphaamylase, catalytic domain PF00128 3.30E−08 241 55 2-Hacid_dh_C 119, 309D-isomer specific 2-hydroxyacid PF02826 1.70E−100 dehydrogenase, NADbinding domain 241 55 2-Hacid_dh 16, 113 D-isomer specific 2-hydroxyacidPF00389 1.50E−23 dehydrogenase, catalytic domain 243 185 PGAM 2, 226Phosphoglycerate mutase family PF00300 4.60E−117 245 271 Ldh_1_N 8, 147lactate/malate dehydrogenase, NAD binding PF00056 9.40E−76 domain 245271 Ldh_1_C 149, 317 lactate/malate dehydrogenase, alpha/beta PF028662.00E−75 C-terminal domain 247 698 Gp_dh_C 157, 318 Glyceraldehyde3-phosphate PF02800 9.70E−88 dehydrogenase, C-terminal domain 247 698Gp_dh_N 3, 156 Glyceraldehyde 3-phosphate PF00044 1.10E−82dehydrogenase, NAD binding domain 249 699 PGK 1, 403 Phosphoglyceratekinase PF00162 1.20E−218 251 752 PGI 7, 442 Phosphoglucose isomerasePF00342 3.80E−136 253 889 Enolase_C 139, 427 Enolase, C-terminal TIMbarrel domain PF00113 2.30E−126 253 889 Enolase_N 5, 135 Enolase,N-terminal domain PF03952 1.40E−58 255 956 PFK 2, 277Phosphofructokinase PF00365 1.70E−174 257 957 PK 1, 346 Pyruvate kinase,barrel domain PF00224 4.30E−228 257 957 PK_C 360, 475 Pyruvate kinase,alpha/beta domain PF02887 2.20E−64 257 957 PEP-utilizers 490, 579PEP-utilising enzyme, mobile domain PF00391 2.50E−32 259 1599F_bP_aldolase 4, 285 Fructose-bisphosphate aldolase class-II PF011167.40E−97 261 1641 SBP_bac_1 7, 343 Bacterial extracellularsolute-binding protein PF01547 1.70E−27 263 452 PTSIIB_sorb 169, 319 PTSsystem sorbose subfamily IIB PF03830 6.20E−76 component 263 452 EIIA-man2, 120 PTS system fructose IIA component PF03610 1.60E−47 265 1479 PRD76, 164; PRD domain PF00874 3.00E−39 181, 275 265 1479 CAT_RBD 2, 60 CATRNA binding domain PF03123 3.10E−16 267 725 PTS_EIIA_1 516, 620phosphoenolpyruvate-dependent sugar PF00358 9.10E−60 phosphotransferasesystem, EIIA 1 267 725 PTS_EIIC 111, 406 Phosphotransferase system, EIICPF02378 1.40E−33 267 725 PTS_EIIB 9, 43 phosphotransferase system, EIIBPF00367 2.00E−16 269 1369 PTS_EIIC 31, 336 Phosphotransferase system,EIIC PF02378 1.30E−60 271 227 PTS_EIIC 26, 364 Phosphotransferasesystem, EIIC PF02378 1.40E−82 273 502 SBP_bac_1 6, 344 Bacterialextracellular solute-binding protein PF01547 5.30E−35 277 1483 ABC_tran28, 215; ABC transporter PF00005 6.40E−88 276, 468 279 1484 RbsD_FucU 1,131 RbsD/FucU transport protein family PF05025 5.90E−59 281 552 Sugar_tr8, 111 Sugar (and other) transporter PF00083 1.80E−09 289 1012PTS_EIIA_1 25, 129 phosphoenolpyruvate-dependent sugar PF00358 8.70E−45phosphotransferase system, EIIA 1 289 1012 PTS_EIIC 278, 562Phosphotransferase system, EIIC PF02378 5.10E−40 289 1012 PTS_EIIB 173,207 phosphotransferase system, EIIB PF00367 5.40E−16 291 1014Alpha-amylase 11, 413 Alpha amylase, catalytic domain PF00128 1.00E−112293 1440 BPD_transp_1 69, 273 Binding-protein-dependent transport systemPF00528 2.40E−27 inner membrane component 295 1442 SBP_bac_1 8, 337Bacterial extracellular solute-binding protein PF01547 2.90E−45 297 1132ABC_tran 342, 523 ABC transporter PF00005 5.60E−35 299 1358 ABC_tran344, 525 ABC transporter PF00005 2.60E−35 301 1838 ABC_tran 33, 218 ABCtransporter PF00005 2.40E−56 305 1913 ABC_tran 33, 214 ABC transporterPF00005 3.10E−57 307 1938 DUF218 177, 331 DUF218 domain PF02698 1.10E−45

Example 3 Sugar Metabolism Genes

Lactobacillus acidophilus has the ability to utilize a variety ofcarbohydrates, including mono-, di- and poly-saccharides, as shown byits API50 sugar fermentation pattern. In particular, complex dietarycarbohydrates that escape digestion in the upper GI-tract, such asraffinose and fructooligosaccharides (Gibson et al. (1995) J. Nutr.125:1401-1412; Barrangou et al. (2003) Proc. Nail. Acari Sci. U, S. A100:8957-8962) can be utilized. The NCFM genome encodes a large varietyof genes related to carbohydrate utilization, including 20phosphoenolpyruvate sugar-transferase systems (PTS) and 5 ATP bindingcassette (ABC) families of transporters. Putative PTS transporters wereidentified for trehalose (ORF 1012) (SEQ ID NOS:103 and 289), fructose(ORF 1777) (SEQ ID NO:35), sucrose (ORF 401) (SEQ ID NO:101), glucoseand mannose (ORF 452 (SEQ ID NOS:1 and 263), ORF 453 (SEQ ID NO:161),ORF 454 (SEQ ID NO:163), ORF 455 (SEQ ID NO:165) and ORF 456 (SEQ IDNO:167)), melibiose (ORF 1705) (SEQ ID NO:33), gentiobiose andcellobiose (ORF 1369) (SEQ ID NOS:17 and 269), salicin (ORF 876 (SEQ IDNO:169), ORF 877 (SEQ ID NO:3), ORF 879 (SEQ ID NO:171)), arbutin (ORF884) (SEQ ID NO:27), and N-acetyl glucosamine (ORF 146) (SEQ ID NO:21).Putative ABC transporters were identified for FOS(ORF 502 (SEQ ID NOS:39and 273) ORF 504 (SEQ ID NO:43), ORF 506 (SEQ ID NO:47)), raffinose (ORF1439 (SEQ ID NO:109), ORF 1440 (SEQ ID NOS:111 and 293), ORF 1441 (SEQID NO:113), ORF 1442 (SEQ ID NOS:115 and 295), and maltose (ORF 1854-ORF1857). A putative lactose-galactose permease was also identified (ORF1463) (SEQ ID NO:175). Most of these transporters share a genetic locuswith a glycosidase and a transcriptional regulator, allowing localizedtranscriptional control.

In silico analyses of the genome revealed the presence of genesrepresenting the complete glycolysis pathway. Additionally, members ofthe general carbohydrate utilization regulation network were identified,namely HPr (ORF 639 (SEQ ID NO:177), ptsH), EI (ORF 640 (SEQ ID NO:179),ptsI), CcpA (ORF 431 (SEQ ID NO:181), ccpA), and HPrK/P (ORF 676 (SEQ IDNO:183), ptsK), indicating an active carbon catabolite repressionnetwork based on sugar availability.

Example 4 Differentially Expressed Genes

Global gene expression patterns obtained from growth on eight differentcarbohydrates were visualized by cluster analysis (Eisen et al. (1998)Proc. Natl. Acad. Sci. USA 95:14863-14868) using Ward's hierarchicalclustering method, volcano plots and contour plots. Overall, between 23and 379 genes were differentially expressed between paired treatmentconditions (with p-values below the Bonferroni correction), representingbetween 1% and 20% of the genome, respectively. All possible treatmentcomparisons were considered, and a gene was considered induced above aparticular level if it showed induction in at least one treatmentcomparison. For genes that showed induction in more than one instance,the highest induction level was selected. Although 342 genes (18% of thegenome) showed induction levels above two fold, only 63 genes (3% of thegenome) showed induction above 4 fold, indicating a relatively smallnumber of genes were highly induced. Although overall expression levelsof the majority of the genes remained consistent regardless of thegrowth substrate (80% of the genome), select clusters showeddifferential transcription of genes and operons. Nevertheless, for eachsugar, a limited number of genes showed specific induction.

In the presence of glucose, ORF 1679 (SEQ ID NO:133) and ORF 1680 (SEQID NO:135) were highly induced when compared to other monosaccharides(fructose, galactose) and di-saccharides (sucrose, lactose, trehalose).The induction levels compared to other sugars varied between 3.5 and 6.3for ORF 1679 (SEQ ID NO:133) and between 3.7 and 4.7 for ORF 1680 (SEQID NO:135). ORF 1679 (SEQ ID NO:133) encodes an ABC nucleotide bindingprotein, including commonly found nucleotide binding domain motifs,namely WalkerA, WalkerB, ABC signature sequence and Linton and Higginsmotif. ORF 1680 (SEQ ID NO:135) encodes an ABC permease, with 10predicted membrane spanning domains. No solute binding protein isencoded in their vicinity, suggesting a possible role as an exporterrather than an importer. Several genes and operons were specificallyrepressed by glucose, including ORFs 680 (SEQ ID NO:239)-ORF 686, whichare involved in glycogen metabolism. Since glycogen is metabolized bythe cell in order to store energy, in the presence of the preferredcarbon source such as glucose, energy storage is not necessary. Othergenes repressed in the presence of glucose included proteins involved inuptake of alternative carbohydrate sources, and enzymes involved inhydrolysis of such carbohydrates.

The three genes of the putative fructose locus, ORF 1777 (SEQ ID NO:35)(FruA, fructose PTS transporter EIIABC^(Fru)), ORF 1778 (SEQ ID NO:185)(FruK, phosphofructokinase EC 2.7.1.56) and ORF 1779 (SEQ ID NO:187)(FruR, transcription regulator) were differentially expressed. Inductionlevels were up to 3.9, 4.3 and 4.6 for fruA, fruK and fruR,respectively. These results suggest fructose is transported into thecell via a PTS transporter, into fructose-6-phosphate, which thephosphofructokinase FruK phosphorylates into fructose-1,6 bi-phosphate,a glycolysis intermediate.

In the presence of sucrose, the three genes of the sucrose locus weredifferentially expressed, namely ORF 399 (SEQ ID NO:97) (ScrR,transcription regulator), ORF 400 (SEQ ID NO:99) (ScrB,sucrose-6-phosphate hydrolase EC 3.2.1.26), and ORF 401 (SEQ ID NO:101)(ScrA, sucrose PTS transporter EIIBCA^(Suc)). When compared to glucose,induction levels were up to 3.1, 2.8 and 17.2 for scrR, scrB and scrA,respectively. ORF 401 (SEQ ID NO:101) in particular showed highinduction levels, between 8.0 and 17.2 when compared to mono- anddi-saccharides. These results indicate that sucrose is transported intothe cell via a PTS transporter, into sucrose-6-phosphate, which issubsequently hydrolyzed into glucose-6-phosphate and fructose by ScrB.

The six genes of the FOS operon were differentially expressed, namelyORF 502 (SEQ ID NOS:39 and 273), ORF 503 (SEQ ID NO:41), ORF 504 (SEQ IDNO:43), ORF 506 (SEQ ID NO:47) (MsmEFGK ABC transporter), ORF 505 (SEQID NO:45) (BfrA, (3-fructosidase EC 3.2.1.26) and ORF 507 (SEQ ID NOS:49and 275) (GtfA, sucrose phosphorylase EC 2.7.1.4). Induction levelsvaried between 15.1 and 40.6 when compared to mono- and di-saccharides,and between 5.5 and 8.9 when compared to raffinose. These resultssuggest FOS is transported into the cell via an ABC transporter andsubsequently hydrolyzed into fructose and sucrose by the fructosidase.Sucrose is likely subsequently hydrolyzed into fructose and glucose-1-Pby the sucrose phosphorylase. In addition to the FOS operon, FOS alsoinduced the fructose operon, the sucrose PTS transporter, the trehaloseoperon and an ABC transporter (ORF 1679-ORF 1680) (SEQ ID NOS:133 and135, respectively).

In the presence of raffinose, the six genes of the raffinose operon werespecifically induced. The raffinose locus consists of ORF 1442 (SEQ IDNOS:115 and 295), ORF 1441 (SEQ ID NO:113), ORF 1440 (SEQ ID NOS:111 and293), ORE 1439 (SEQ ID NO:109) (MsmEFGK₂ ABC transporter), ORF 1438 (SEQID NO:197) (MelA α-galactosidase EC 3.2.1.22), and ORF 1437 (SEQ IDNO:195) (GtfA₂, sucrose phosphorylase EC 2.7.1.4). Induction levelsvaried between 15.1 and 45.6, when compared to all other conditions.Additionally, ORFs 1433 (SEQ ID NO:189), 1434 (SEQ ID NO:191)(di-hydroxyacetone kinase EC 17.129), and ORF 1436 (SEQ ID NO:193)(glycerol uptake facilitator) were induced between 1.9 and 24.7 foldwhen compared to other conditions.

In the presence of lactose and galactose, ten genes distributed in twoloci were differentially expressed, namely ORF 1463 (SEQ ID NO:175)(LacS permease of the GPH translocator family), ORF 1462 (SEQ ID NO:209)(LacZ, β-galactosidase EC 3.2.1.23), ORF 1461 (SEQ ID NO:207), ORF 1460(SEQ ID NO:205)(surface protein), ORF 1459 (SEQ ID NO:203) (GalK,galactokinase EC 2.7.1.6), ORF 1458 (SEQ ID NO:201)(GalT, galactose-1phosphate uridylyl transferase EC 2.7.7.10), ORF 1457 (SEQ IDNO:199)(GalM, galactose epimerase EC 5.1.3.3), ORFs 1467 (SEQ IDNO:211), 1468 (SEQ ID NO:213)(LacLM, β-galactosidase EC 3.2.1.23 largeand small subunits), and 1469 (SEQ ID NO:215)(GalE, UDP-glucoseepimerase EC 5.1.3.2). LacS (SEQ ID NO:175) is similar to GPH permeasespreviously identified in lactic acid bacteria. Although LacS (SEQ IDNO:175) contains an EIIA at the carboxy-terminus, it is not a PTStransporter. Also, LacS (SEQ ID NO:175) includes a His at position 553,which might be involved in interaction with HPr, as shown in S.salivarius (Lessard et al. (2003) J. Bacteriol. 185:6764-6772). In thepresence of lactose and galactose, galKTM (SEQ ID NOS:199, 201, and 203)were induced between 3.7 and 17.6 fold; lacSZ (SEQ ID NOS:175 and 209)were induced between 2.8 and 17.6 fold; lacL (SEQ ID NO:213) and galE(SEQ ID NO:215) were induced between 2.7 and 29.5, when compared toother carbohydrates not containing galactose, i.e., glucose, fructose,sucrose, trehalose and FOS. These results suggest lactose is transportedinto the cell via the LacS permease of the galactoside-pentosehexuronide translocator family. Inside the cell, lactose is hydrolyzedinto glucose and galactose by LacZ. Galactose is then phosphorylated byGalK into galactose-1 phosphate, further transformed into UDP-galactoseby GalT. UDP-galactose is subsequently epimerized to UDP-glucose byGalE. UDP-glucose is likely turned into glucose-1P by ORF 1719 (SEQ IDNO:217), which encodes a UDP-glucose phosphorylase EC 2.7.7.9,consistently highly expressed. Finally, the phosphoglucomutase EC5.4.2.2 likely acts on glucose-1P to yield glucose-6P, a glycolysissubstrate.

The three genes of the putative trehalose locus were also differentiallyexpressed. The trehalose locus consists of ORF 1012 (SEQ ID NOS:103 and289)(encoding the TreB trehalose PTS transporter EIIABC^(Tre) EC23.1.69), ORF 1013 (SEQ ID NO:105)(TreR, trehalose regulator) and ORF1014 (SEQ ID NOS:107 and 291) (TreC, trehalose-6 phosphate hydrolase EC3.2.1.93). Induction levels were between 4.3 and 18.6 for treB (SEQ IDNOS:103 and 289), between 2.3 and 7.3 for treR (SEQ ID NO:105), andbetween 2.7 and 18.5 for treC, (SEQ ID NOS:107 and 291), when comparedto glucose, sucrose, raffinose and galactose. These results suggesttrehalose is transported into the cell via a PTS transporter,phosphorylated to trehalose-6 phosphate and hydrolyzed into glucose andglucose-6 phosphate by TreC.

In addition, genes showing differential expression included sugar- andenergy-related genes ORF 874 (SEQ ID NO:219) (beta galactosidase EC3.2.1.86), ORF 910 (SEQ ID NO:221) (L-LDH EC 1.1.1.27), ORF 1007 (SEQ IDNO:223 (pyridoxal kinase 2.7.1.35), ORF 1812 (SEQ ID NO:225) (alphaglucosidase EC 3.2.1.3), ORF 1632 (SEQ ID NO:227) (aldehydedehydrogenase EC 1.2.1.16), ORF 1401 (SEQ ID NO:229) (NADH peroxidase EC1.11.1.1), ORF 1974 (SEQ ID NO:231) (pyruvate oxidase EC 1.2.3.3),adherence genes ORF 555, ORF 649, ORF 1019; aminopeptidase ORF 911, ORF1086; amino-acid permease, ORF 1102 (SEQ ID NO:233) (membrane protein),ORF 1783 (SEQ ID NO:235) (ABC transporter), and ORF 1879 (SEQ IDNO:237)(pyrimidine kinase EC 2.7.4.7).

Example 5 Real Time RT-PCR

Five genes that were differentially expressed in microarray experimentswere selected for real-time quantitative RT-PCR experiments, in order tovalidate induction levels measured by microarrays. These genes wereselected for both their broad expression range (LSM between −1.52 and+3.87), and induction levels between sugars (fold induction up to 34).All selected genes showed an induction level above 6 fold in at leastone instance. Also, the annotations of the selected genes werecorrelated functionally with carbohydrate utilization. The five selectedgenes were: beta-fructosidase (ORF 505) (SEQ ID NO:45), trehalose PTS(ORF 1012) (SEQ ID NOS:103 and 289), glycerol uptake facilitator (ORF1436) (SEQ ID NO:193), beta-galactosidase (ORF 1467) (SEQ ID NO:211),and ABC transporter (ORF 1679) (SEQ ID NO:133).

For the five selected genes, induction levels were compared between sixdifferent treatments, resulting in 15 induction levels for each gene.The induction levels measured by microarrays were plotted againstinduction levels measured by Q-PCR, in order to validate microarraydata. Individual R-square values ranged between 0.642 and 0.883 for eachof the tested genes (between 0.652 and 0.978 using data in a log₂scale). When the data were combined, the global R-square value was 0.78(0.88 using data in a log₂ scale). A correlation analysis was run in SAS(Cary, N.C.), and showed a correlation between the two methods withP-values less than 0.001, for Spearman, Hoeffding and Kendall tests.Additionally, a regression analysis was run in excel (Microsoft, CA),and showed a statistically highly significant (p<1.02×10⁻²⁵) correlationbetween microarray data and Q-PCR results. Nevertheless, Q-PCRmeasurements revealed larger induction levels, which is likely due tothe smaller dynamic range of the microarray scanner, compared to that ofthe Q-PCR cycler. Similar results have been reported previously (Wagneret al. (2003) J. Bacteriol. 185:2080-2095).

Example 6 Comparative Analysis

Comparative analyses of global transcription profiles determined forgrowth on eight carbohydrates identified the basis for carbohydratetransport and catabolism in L. acidophilus. Specifically, threedifferent types of carbohydrate transporters were differentiallyexpressed, namely phosphoenolpyruvate: sugar phosphotransferase system(PTS), ATP binding cassette (ABC) and galactoside-pentose hexuronide(GPH) translocator, illustrating the diversity of carbohydratetransporters used by Lactobacillus acidophilus. Transcription profilessuggested that galactosides were transported by a GPH translocator,while mono- and di-saccharides were transported by members of the PTS,and polysaccharides were transported by members of the ABC family.

Microarray results indicated fructose, sucrose and trehalose aretransported by PTS transporters EIIABC^(Fru) (ORF 1777) (SEQ ID NO:35),EIIBCA^(Suc) (ORF 401) (SEQ ID NO:101) and ElIABC^(Tre) (ORF 1012) (SEQID NOS:103 and 289), respectively. Those genes are encoded on typicalPTS loci (FIG. 2), along with regulators and enzymes that have been wellcharacterized in other organisms. In contrast, FOS and raffinose aretransported by ABC transporters of the MsmEFGK family, ORFs 502 (SEQ IDNOS:39 and 273), 503 (SEQ ID NO:41), 504 (SEQ ID NO:43), and 505 (SEQ IDNO:45); and ORFs 1437 (SEQ ID NO: 195, ORF 1438 (SEQ ID NO:197), 1439(SEQ ID NO:109), ORF 1440 (SEQ ID NOS:111 and 293), ORF 1441 (SEQ IDNO:113), and ORF 1442 (SEQ ID NO:115 and 295), respectively. In the caseof trehalose and FOS, microarray results correlate well with functionalstudies in which targeted knock out of carbohydrate transporters andhydrolases modified the saccharolytic potential of Lactobacillusacidophilus NCFM. Differential expression of the EIIABC^(Tre) isconsistent with recent work in Lactobacillus acidophilus indicating ORF1012 (SEQ ID NOS:103 and 289) is involved in trehalose uptake.Similarly, differential expression of the fos operon is consistent withprevious work in Lactobacillus acidophilus indicating those genes areinvolved in uptake and catabolism of FOS, and induced in the presence ofFOS and repressed in the presence of glucose (Barrangou et al. (2003)Proc. Natl. Acad. Sci. USA 100: 8957-8962). Additionally, induction ofthe raffinose msm locus is consistent with previous work inStreptococcus mutans (Russell et al. (1992) J. Biol. Chem. 267:4631-4637) and Streptococcus pneumoniae (Rosenow et al. (1999) GenomeRes. 9:1189-1197).

A number of lactic acid bacteria take up glucose via a PTS transporter.The EII^(Man) PTS transporter has the ability to import both mannose andglucose (Cochu et al. 2003). The Lactobacillus acidophilus mannose PTSsystem is similar to that of Streptococcus thermophilus, with proteinssharing 53-65% identity and 72-79% similarity. Specifically, theEII^(Man) is composed of three proteins IIAB^(Man), IIc^(Man) andIID^(Man), encoded by ORF 452 (SEQ ID NOS:1 and 263) (manL), ORF 455(SEQ ID NO:165) (manM) and ORF 456 (SEQ ID NO:167) (manN), respectively(FIG. 2). Most of the carbohydrates examined here specifically inducedgenes involved in their own transport and hydrolysis, but glucose didnot. Analysis of the mannose PTS revealed that the genes encoding theEHABCD^(Man) were consistently highly expressed, regardless of thecarbohydrate source. This expression profile suggests glucose is apreferred carbohydrate, and Lactobacillus acidophilus is also designedfor efficient utilization of different carbohydrate sources, assuggested previously for Lactobacillus plantarum (Kleerebezem et al,(2003) Proc. Natl. Acad. Sci. USA 100:1990-1995).

The genes differentially expressed in the presence of galactose andlactose included a permease (LacS), and the enzymatic machinery of theLeloir pathway. Members of the LacS subfamily ofgalactoside-pentose-hexuronide (GPH) translocators have been describedin a variety of lactic acid bacteria, including Leuconostoc lactis(Vaughan et al. (1996) Appl. Environ. Microbiol. 62:1574-1582), S.thermophilus (van den Bogaard et al. (2000) J. Bacteriol.182:5982-5989), Streptococcus salivarius (Lessard et al. (2003) J.Bacteriol. 185:6764-6772) and Lactobacillus delbrueckii (Lapierre et al.(2002) J. Bacteriol. 184:928-935). Although LacS contains a PTS EIIA atthe carboxy terminus, it is not a member of the PTS family oftransporters. LacS has been reported to have the ability to import bothgalactose and lactose in select organisms (Vaughan et al. (1996) Appl.Environ. Microbiol. 62:1574-1582; van den Bogaard et al. (2000) J.Bacteriol. 182:5982-5989). Although the combination of a LacS lactosepermease with two β-galactosidasesubunits LacL and LacM has beendescribed in Lactobacillus plantarum (Kleerebezem et al. 2003) andLeuconostoc lactis (Vaughan et al. (1996) Appl. Environ. Microbiol.62:1574-1582), it has never been reported in Lactobacillus acidophilus.Even though constitutive expression of lacS and lacLM has been reportedpreviously (Vaughan et al. (1996) Appl. Environ. Microbiol.62:1574-1582), these results indicate specific induction of the genesinvolved in uptake and catabolism of both galactose and lactose. Operonorganization for galactoside utilization is variable and unstable amongGram-positive bacteria (Lapierre et al. (2002) J. Bacteriol.184:928-935; Vaillancourt et al. (2002) J. Bacteriol. 184:785-793;Boucher et al. (2003) Appl. Environ. Microbiol. 69:4149-4156; Fortina etal. (2003) Appl. Environ. Microbiol. 69:3238-3243; Grossiord et al.(2003) J. Bacteriol. 185:870-878). Even amongst closely relatedLactobacillus species, namely Lactobacillus johnsonii, Lactobacillusgasseri and Lactobacillus acidophilus, the lactose-galactose locus isnot well conserved (Pridmore et al. (2004) Proc. Natl. Acad. Sci. USA101:2512-2517).

Although it was previously suggested that the phosphoenolpyruvate:phosphotransferase system is the primary sugar transport system ofGram-positive bacteria (Ajdic et al. (2002) Proc. Natl. Acad. Sci. USA99:14434-14439; Warner and Lolkema (2003) Microbiol. Mol. Rev.67:475-490), current microarray data indicate that ABC transport systemsare also important. While PTS transporters are involved in uptake ofmono- and di-saccharides, those carbohydrates are digested in the upperGIT. In contrast, oligosaccharides reach the lower intestine wherebycommensals are likely to compete for more complex and scarce nutrients.Perhaps under such conditions ABC transporters are even more crucialthan the PTS, given their apparent roles in transport ofoligosaccharides like FOS and raffinose. In this regard, the ability toutilize nutrients that has been are non digestible by the host has beenassociated with competitiveness and persistence of beneficial intestinalflora in the colon (Schell et al. (2002) Proc. Natl. Acad. Sci. USA99:14422-14427).

Transcription profiles of genes differentially expressed in conditionstested indicated that all carbohydrate uptake systems and theirrespective sugar hydrolases were specifically induced by theirsubstrate, except for glucose. Moreover, genes within those inducibleloci were repressed in the presence of glucose, and cre sequences wereidentified in their promoter-operator regions. Together, these resultsindicate regulation of carbohydrate uptake and metabolism at thetranscription level, and implicate the involvement of a globalregulatory system compatible with carbon catabolite repression. Carboncatabolite repression (CCR) controls transcription of proteins involvedin transport and catabolism of carbohydrates (Miwa et al. (2000) NucleicAcids Res. 28:1206-1210). Catabolite repression is a mechanism widelydistributed amongst Gram-positive bacteria, mediated in cis bycatabolite responsive elements (Miwa et al. (2000) Nucleic Acids Res.28:1206-1210; Wickert and Chambliss (1990) Proc. Natl. Acad. Sci. USA87:6238-6242), and in trans by repressors of the Lad family, which isresponsible for transcriptional repression of genes encoding unnecessarysaccharolytic components in the presence of preferred substrates(Wickert and Chambliss (1990) Proc. Natl. Acad. Sci. USA 87:6238-6242;Viana et al. (2000) Mol. Microbiol. 36:570-584; Muscariello et al.(2001) Appl. Environ. Microbiol. 67:2903-2907; Warner and Lolkema (2003)Microbiol. Mol. Rev. 67:475-490). This regulatory mechanism allows cellsto coordinate the utilization of diverse carbohydrates, to focusprimarily on preferred energy sources. CCR is based upon several keyenzymes, namely HPr (ORF 639 (SEQ ID NO:177), ptsH), EI (ORF 640 (SEQ IDNO:179), ptsI), CcpA (ORF 431 (SEQ ID NO:181), ccpA), and HPrK/P (ORF676 (SEQ ID NO:183), ptsK), all of which are encoded within theLactobacillus acidophilus chromosome.

Carbon catabolite repression has already been described in lactobacilli(Mahr et al. 2000). The PTS is characterized by a phosphate transfercascade involving PEP, EI, HPr, EIIABC, whereby a phosphate isultimately transferred to the carbohydrate substrate (Saier, 2000;Warner and Lolkema, 2003). HPr is an important component of CCR, whichis regulated via phosphorylation by enzyme I and HPrK/P. When HPr isphosphorylated at His15, the PTS is active, and carbohydratestransported via the PTS are phosphorylated via EIIABCs. In contrast,when HPr is phosphorylated at Ser46, the PTS machinery is not functional(Mijakovic et al. (2002) Proc. Natl. Acad, Sci. USA 99:13442-13447).

Although the phosphorylation cascade suggests regulation at the proteinlevel, several studies report transcriptional modulation of ccpA andptsHI. In S. thermophilus, CcpA production is induced by glucose (vanden Bogaart et al. 2000). In several bacteria, the carbohydrate sourcemodulates ptsHI transcription levels (Luesink et al. 1999). In contrast,expression levels of ccpA, ptsH, ptsI and ptsK did not vary in thepresence of different carbohydrates in Lactobacillus acidophilus. Theseresults are consistent with regulation via phosphorylation at theprotein level. Similar results have been reported for ccpA expressionlevels in Lactobacillus pentosus (Mahr et al. (2000) Appl. Environ.Microbiol. 66:277-283), and ptsHI transcription in S. thermophilus(Cochu et al. (2003) Appl. Environ. Microbiol. 69:5423-5432).

Globally, microarray results allowed reconstruction of carbohydratetransport and catabolism pathways (FIG. 2). Although transcription ofcarbohydrate transporters and hydrolases was specifically induced bytheir respective substrates, these glycolysis genes were consistentlyhighly expressed: D-lactate dehydrogenase (D-LDH, ORF 55 (SEQ IDNO:241)), phosphoglycerate mutase (PGM, ORF 185 (SEQ ID NO:243)),L-lactate dehydrogenase (L-LDH, ORF 271 (SEQ ID NO:245)), glyceraldehyde3-phosphate dehydrogenase (GPDH, ORF 698 (SEQ ID NO:247)),phosphoglycerate kinase (PGK ORF 699 (SEQ ID NO:249)), glucose6-phosphate isomerase (GPI, ORF 752 (SEQ ID NO:251)), 2-phosphoglyceratedehydratase (PGDH, ORF 889 (SEQ ID NO:253)), phosphofructokinase (PFK,ORF 956 (SEQ ID NO:255)), pyruvate kinase (PK, ORF 957 (SEQ ID NO:257))and fructose biphosphate aldolase (FBPA, ORF 1599 (SEQ ID NO:259)). Aglycerol-3-phosphate ABC transporter (ORF 1641 (SEQ ID NO:261)) was alsoamong the genes that were consistently highly expressed. Orchestratedcarbohydrate uptake likely withdraws energy sources from the intestinalenvironment and deprives other bacteria of access to such resources.Consequently, Lactobacillus acidophilus may compete well against othercommensals for nutrients.

In summary, a variety of carbohydrate uptake systems were identified andcharacterized, with respect to expression profiles in the presence ofdifferent carbohydrates, including PTS, ABC and GHP transporters. Theuptake and catabolic machinery is highly regulated at the transcriptionlevel, suggesting the Lactobacillus acidophilus transcriptome isflexible, dynamic and designed for efficient carbohydrate utilization.Differential gene expression indicated the presence of a global carboncatabolite repression regulatory network. Regulatory proteins wereconsistently highly expressed, suggesting regulation at the proteinlevel, rather than the transcriptional level. Collectively,Lactobacillus acidophilus appears to be able to efficiently adapt itsmetabolic machinery to fluctuating carbohydrate sources available in thenutritional complex environment of the small intestine. In particular,ABC transporters of the MsmEFG family involved in uptake of FOS andraffinose likely play an important role in the ability of Lactobacillusacidophilus to compete with intestinal commensals for complex sugarsthat are not digested by the human host. Ultimately, this informationprovides new insights into how undigested dietary compounds influencethe intestinal microbial balance. This study is a model for comparativetranscriptional analysis of a bacterium exposed to varying growthsubstrates.

Example 7 Multidrug Transporters

Microorganisms such as Lactobacillus acidophilus have developed variousmethods in which to resist the toxic effect of antibiotics and otherdeleterious compounds. One such method involves transporters thatpromote the active efflux of drugs, by which drug resistance may beaffected for a particular microorganism. There are two major classes ofmultidrug transporters: secondary multidrug transporters that utilizethe transmembrane electrochemical gradient of protons or sodium ions todrive the extrusion of drugs from a cell; and ATP-binding cassette(ABC)-type multidrug transporters that utilize the free energy of ATPhydrolysis to pump drugs out of the cell.

Secondary multidrug transporters are subdivided into several distinctfamilies of transport proteins: the major facilitator superfamily (MFS,Pao et al. (1998) Microbiol. Mol. Biol. Rev. 62:1-34), the smallmultidrug resistance (SMR) family (Paulsen et al. (1996) Mol. Microbiol.19:1167-1175), the resistance-nodulation-cell division (RND) family(Saier et al. (1994) Mol. Microbiol. 11:841-847), and the multidrug andtoxic compound extrusion (MATE) family (Brown et al. (1999) Mol.Microbiol. 31:394-395. These families are not solely associated withmultidrug export, and include proteins involved in other proton motiveforce-dependent transport processes or other functions.

MFS membrane transport proteins are involved in synport, antiport, oruniport of various substrates, among which are antibiotics (Marger andSaier (1993) Trends Biochem. Sci. 18:13-20). Analysis and alignment ofconserved motifs of the resistance-conferring drug efflux proteinsrevealed that these proteins can be divided into two separate clusters,with either 12 or 14 transmembrane segments (Paulsen and Skurry (1993)Gene 124:1-11). The NCFM genome contains several genes that encode MFStransporters attributed to multidrug transport. Included among these arethe transporters encoded in ORFs 552 (SEQ ID NO:77), 566 (SEQ ID NO:79),567 (SEQ ID NO:81), 1446 (SEQ ID NO:85), 1471 (SEQ ID NO:87), 1621 (SEQID NO:91), 1853 (SEQ ID NO:93), 1854 (SEQ ID NO:321), 164 (SEQ IDNO:309), 251-253 (SEQ ID NOs:311, 313, 315) and 1062 (SEQ ID NO:317).

ABC transporters require four distinct domains: two hydrophobic membranedomains, which usually consist of six putative transmembrane α-heliceseach, and two hydrophilic nucleotide binding domains (NBDs), containingWalker A and B motifs (Walker et al. (1982) EMBO J. 1:945-951) and theABC signature (Hyde et al. (1990) Nature 346:362-365). The individualdomains can be expressed as separate proteins or they may be fused intomultidomain polypeptides in several ways (Faith and Kolter (1993)Microbiol. Rev. 57:995-1017; Higgens (1992) Annu. Rev. Cell Bio.8:67-113; Hyde et al. (1990) Nature 346:362-365). A multidrug ABCtransporter in the NCFM genome similar to the ABC multidrug transporterlmrA from Lactococcus lactis and horA from Lactobacillus brevis isencoded by ORF 597.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule comprising a nucleotide sequence as setforth in SEQ ID NO: 115 or 117; b) a nucleic acid molecule comprising anucleotide sequence having at least 90% sequence identity to thenucleotide sequence as set forth in SEQ ID NO: 115 or 117, wherein saidnucleic acid molecule encodes a polypeptide having biological activity;c) a nucleic acid molecule comprising a fragment of a nucleotidesequence as set forth in SEQ ID NO: 115 or 117, wherein said nucleicacid molecule encodes a polypeptide having biological activity; d) anucleic acid molecule comprising a nucleotide sequence encoding apolypeptide comprising an amino acid sequence as set forth in SEQ ID NO:116 or 118; e) a nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide comprising a fragment of an amino acid sequenceas set forth in SEQ ID NO 116 or 118, wherein said polypeptide hasbiological activity; and, f) a nucleic acid molecule comprising anucleotide sequence encoding a polypeptide comprising an amino acidsequence having at least 90% sequence identity with an amino acidsequence as set forth in SEQ ID NO: 116 or 118, wherein said polypeptidehas biological activity.
 2. A plasmid comprising the nucleic acidmolecule of claim
 1. 3. The plasmid of claim 2, further comprising asecond nucleic acid molecule encoding a heterologous polypeptide.
 4. Amicrobial host cell comprising the plasmid of claim
 2. 5. The microbialhost cell of claim 4, wherein said microbial host cell is a bacterialhost cell.
 6. The bacterial host cell of claim 5, wherein said bacterialhost cell comprises a lactic acid bacterium or Lactobacillusacidophilus.
 7. A method for producing a polypeptide, comprisingculturing a microbial cell comprising a heterologous nucleic acidmolecule encoding a polypeptide comprising an amino acid sequence havingat least 90% sequence identity with the amino acid sequence of SEQ IDNO: 116 or 118 and culturing said cell under conditions in which saidheterologous nucleic acid molecule is expressed.
 8. An antibody thatselectively binds to the polypeptide of claim
 1. 9. The isolated nucleicacid molecule of claim 1, wherein said nucleic acid molecule comprises anucleotide sequence having at least 90% sequence identity to thenucleotide sequence of SEQ ID NO: 115 or
 117. 10. The isolated nucleicacid molecule of claim 1, wherein said nucleic acid molecule comprisesthe nucleotide sequence of SEQ ID NO: 115 or
 117. 11. The isolatednucleic acid molecule of claim 1, wherein said nucleic acid moleculecomprises a nucleotide sequence that encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 116 or
 118. 12. The plasmid ofclaim 2, wherein said nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO: 115 or
 117. 13. The plasmid of claim 2, whereinsaid nucleic acid molecule comprises a nucleotide sequence having atleast 90% sequence identity to the nucleotide sequence of SEQ ID NO: 115or
 117. 14. The plasmid of claim 2, wherein said nucleic acid moleculecomprises a nucleotide sequence that encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 116 or
 118. 15. The microbial hostcell of claim 4, wherein said nucleic acid molecule comprises thenucleotide sequence of SEQ ID NO: 115 or
 117. 16. The microbial hostcell of claim 4, wherein said nucleic acid molecule comprises anucleotide sequence having at least 90% sequence identity to thenucleotide sequence of SEQ ID NO: 115 or
 117. 17. The microbial hostcell of claim 4, wherein said nucleic acid molecule comprises anucleotide sequence that encodes a polypeptide comprising the amino acidsequence of SEQ ID NO: 116 or
 118. 18. A microbial host cell comprisinga heterologous nucleic acid molecule that encodes a polypeptidecomprising an amino acid sequence having at least 90% sequence identityto the amino acid sequence of SEQ ID NO: 116 or
 118. 19. The microbialhost cell of claim 18, wherein said microbial host cell is a bacterialhost cell.
 20. The microbial host cell of claim 19, wherein saidbacterial host cell is a lactic acid bacterium.
 21. The microbial hostcell of claim 20, wherein said lactic acid bacterium is Lactobacillusacidophilus.
 22. The microbial host cell of claim 16, wherein saidnucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:115 or
 117. 23. The microbial host cell of claim 18, wherein saidnucleic acid molecule comprises a nucleotide sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 116 or 118.24. The method of claim 7 wherein said nucleic acid molecule comprisesthe nucleotide sequence of SEQ ID NO: 115 or
 117. 25. The method ofclaim 7, wherein said nucleic acid molecule comprises a nucleotidesequence having at least 90% sequence identity to the nucleotidesequence of SEQ ID NO: 115 or
 117. 26. The method of claim 7, whereinsaid nucleic acid molecule comprises a nucleotide sequence that encodesa polypeptide comprising the amino acid sequence of SEQ ID NO: 116 or118.
 27. The method of claim 7, wherein said microbial host cell is abacterial host cell.
 28. The method of claim 27, wherein said bacterialhost cell is a lactic acid bacterium.
 29. The method of claim 28,wherein said lactic acid bacterium is Lactobacillus acidophilus.